基于肠细胞生长及健脾中药效应的RPS20基因功能鉴定
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摘要
研究背景和目的
脾虚证是中医辨证的常见证型,现代医学认为脾虚证的临床表现多与消化吸收和代谢功能异常相关,国内外学者从消化系统、免疫系统、物质能量代谢等角度对脾虚证进行了大量的探索研究,初步阐释脾虚证的现代病理生理学基础。由于中医证候具有系统性和复杂性,局部的研究难以全面地反映脾虚证的本质。近年来,基于基因组学技术的中医证候基因差异表达谱研究,可从整体水平反映中医证候的生物学基础,应用基因芯片等技术对中医证候差异表达基因的探讨分析为阐明中医证候的本质提供了新的思路和方法,并取得了一定的进展。
本实验室在前期课题对慢性浅表性胃炎脾虚证患者胃粘膜基因差异表达谱的研究中发现,脾虚证患者核糖体蛋白S20(RPS20)等基因表达出现下调趋势,并通过荧光定量PCR验证了RPS20等基因的表达下调呈阳性结果。后续对RPS20基因的体外功能研究结果初步表明核糖体蛋白基因在脾虚证发生发展过程中可能具有重要作用,RPS20基因RNA干扰后大鼠小肠上皮细胞(IEC-6)的形态结构发生改变,细胞的迁移、增殖能力受到抑制,提示RPS20基因表达下调可能影响小肠上皮细胞的消化吸收和粘膜损伤修复功能。此外通过小鼠结肠腺癌细胞(CT26)筛选了针对小鼠RPS20基因的RNA干扰有效序列,重组构建慢病毒RNA干扰表达载体,以期在动物体内对RPS20基因进行后续的功能研究。
本研究将在前期研究的基础上进一步开展RPS20基因的功能鉴定,通过考察益气健脾中药对PRS20基因RNA干扰后IEC-6细胞受损的形态和功能的作用,从“药物作用”的角度反证前期的研究结果,进一步探讨研究RPS20的基因功能,以支持课题组前期的研究发现。此外,根据前期构建的RNA干扰表达载体包装RNA干扰慢病毒,考察其转染后对CT26细胞生长的影响,进一步验证前期所筛选的RNA干扰序列的有效性,并为后续在小鼠体内采用RNA干扰技术进行RPS20基因功能研究提供技术参考。
研究方法
1.采用siRNA转染IEC-6细胞对RPS20基因进行RNA干扰,给予益气健脾中药提取物作用后,检测IEC-6细胞的增殖和迁移能力、细胞形态结构、RPS20表达情况和细胞内的多胺含量。
(1)IEC-6细胞生长情况的考察：分别采用细胞计数法和xCELLigence实时细胞分析仪(RTCA)考察IEC-6细胞的生长曲线,为后续实验选择合适的细胞接种密度和实验干预时间点提供参考。
(2)选择RNA干扰的转染条件：在前期合成的siRNA干扰序列的基础上加入荧光表达序列,设置不同的转染条件,采用RTCA检测转染后IEC-6细胞的生长情况,以转染后细胞的生长情况直接反映RNA干扰的效果,并结合转染后细胞的荧光表达情况选择合适的转染条件。
(3)健脾中药对IEC-6细胞增殖能力的作用：采用RTCA检测益气健脾中药黄芪、甘草、党参提取物对正正常IEC-6细胞和RNA干扰后IEC-6细胞增殖能力的影响,并选择作用效果较明显的中药提取物进行后续实验研究。
(4)健脾中药对IEC-6细胞迁移能力的作用：采用IEC-6细胞划痕损伤迁移模型,研究黄芪提取物对正正常IEC-6细胞和RNA干扰后IEC-6细胞在划痕损伤后迁移能力的影响。
(5)健脾中药对IEC-6细胞形态结构的作用：分别采用光学显微镜和透射电镜观察RNA干扰和给予黄芪提取物作用后IEC-6细胞形态和结构的变化情况,考察中药对RNA干扰后IEC-6细胞受损的形态结构的恢复作用。
(6)健脾中药对IEC-6细胞RPS20表达水平的作用：分别采用荧光定量PCR和Westernblot检测RNA干扰和给予黄芪提取物作用后IEC-6细胞中RPS20mRNA和蛋白的表达水平。
(7)健脾中药对IEC-6细胞中多胺含量的影响：对RNA干扰和黄芪提取物作用后的IEC-6细胞进行样品处理,采用苯甲酰氯柱前衍生反相高效液相色谱法检测各样品中精脒的含量。
2.RNA干扰慢病毒对CT26细胞生长的影响：将前期重组构建的针对CT26细胞的慢病毒表达载体与慢病毒包装试剂共转染293FT细胞,包装获得RNA干扰慢病毒,测定病毒滴度并将其转染CT26细胞,采用RTCA检测转染后细胞的生长情况,研究RNA干扰慢病毒对CT26细胞生长的影响。
研究结果
1.RNA干扰IEC-6细胞和益气健脾中药的作用：
(1)IEC-6细胞的生长情况：细胞计数法绘制以1×104cells/well的密度接种于24孔培养板中IEC-6细胞的生长曲线,细胞从接种的第3天开始进入对数生长期,接种后第10天进入缓慢生长的平台期；RTCA检测得到不同接种密度的23代和26代IEC-6细胞的生长曲线,在相同接种密度条件下,23代和26代的IEC-6细胞生长趋势相似,随着接种密度的增大,细胞生长达到平台期的时间缩短。
(2)RNA干扰的转染条件：RTCA检测结果发现高剂量和中剂量的转染试剂(Lipofectamine2000)对细胞生长有明显毒害作用；不同剂量组合的转染混合物在转染初期对IEC-6细胞生长的抑制作用相近,但在相同转染试剂用量时siRNA剂量的增加能延长对细胞生长抑制作用的持续时间。结合荧光表达的情况,最后选择RNA干扰的转染条件为：E-Plate16培养板中每孔加入0.3μL的Lipofectamine2000和16pmol的siRNA(浓度分别为1.5mg/L和80nmol/L)。
(3)健脾中药对IEC-6细胞增殖能力的作用：黄芪和党参提取物能促进正正常IEC-6细胞的增殖,甘草对正常IEC-6细胞的作用不明显；RNA干扰后IEC-6细胞的增殖能力下降,而黄芪、甘草和党参提取物都能有效促进RNA干扰后IEC-6细胞的增殖,其中黄芪的效果较优。结果表明健脾中药可在一定程度上逆转RPS20基因干扰引发的IEC-6细胞增殖能力下降的病理表现。
(4)健脾中药对IEC-6细胞迁移能力的作用：高剂量和中剂量的黄芪提取物能有效促进正常IEC-6细胞在划痕损伤后的迁移能力；RNA干扰后IEC-6细胞的迁移能力显著下降,经黄芪提取物作用后其迁移能力较转染模型组显著提高,但未能恢复到正正常水平。结果表明RPS20基因干扰后IEC-6细胞受损的迁移能力可在健脾中药作用后得到一定程度的恢复。
(5)健脾中药对IEC-6细胞形态结构的作用：光学显微镜观察发现RNA干扰后IEC-6细胞数目减少,细胞形态改变,细胞间隙增大,给予黄芪提取物作用后细胞受损的形态得到不同程度的改善；透射电镜观察发现RNA干扰后IEC-6细胞胞浆中空泡和溶酶体数目增多,线粒体减少,经治疗后细胞受损结构得到一定程度的改善。
(6)健脾中药对IEC-6细胞RPS20表达的作用：RNA干扰后IEC-6细胞中RPS20mRNA表达被显著抑制,给予黄芪提取物作用后其表达量有所提高,其中中剂量组与转染模型组比较具有显著差异性；RPS20蛋白表达在RNA干扰后被显著抑制,但经健脾中药作用后RPS20蛋白表达没有提高。
(7)健脾中药对IEC-6细胞中多胺含量的影响：精脒对照品的回归方程为Y=25.69X—1.337,相关系数R=0.9998,精脒对照品在0.7600～7.600nmol范围内线性良好。检测结果RNA干扰后IEC-6细胞中精脒含量比正正常组增加,但无统计学意义；经黄芪提取物作用后的各组细胞中精脒含量较正正常组显著增加,但与RNA干扰模型组比较无明显差异。
2.RNA干扰慢病毒对CT26细胞生长的影响：慢病毒滴度为1.2×105TU/mL,RNA干扰慢病毒转染使CT26细胞指数的增长受到明显抑制,转染后30h细胞指数抑制率达到82.24%。结果表明根据前期筛选的RNA干扰序列包装获得的慢病毒转染后能有效抑制CT26细胞中RPS20的表达从而抑制细胞的生长。
结论
根据xCELLigence RTCA检测结果和细胞荧光表达情况选择的RNA干扰条件转染IEC-6细胞,可有效抑制IEC-6细胞RPS20的表达,并抑制细胞的增殖和迁移能力,影响细胞的形态结构；给予益气健脾中药黄芪的提取物作用后可提高RNA干扰后IEC-6细胞中RPS20mRNA的表达,促进细胞的增殖和迁移,并在一定程度上改善细胞受损的形态结构；给予益气健脾中药作用后,能改善RPS20表达异常所导致的细胞形态和功能的异常,提示RPS20可能与脾虚证的症状表现之间存在一定联系,本研究从药物作用的角度进一步支持课题组前期对脾虚证差异表达基因的研究发现及对RPS20在IEC-6细胞的生物功能鉴定的研究结果；本研究可为中医证候差异表达基因的功能研究提供参考。
本研究还验证了前期针对CT26细胞重组构建的慢病毒表达载体可成功用于包装RNA干扰慢病毒,以其转染CT26细胞能有效抑制细胞的生长,提示RNA干扰慢病毒可能用于小鼠体内RNA干扰进行RPS20功能鉴定的后续研究。
Background and Objective
Spleen deficiency is a common syndrome of Chinese medicine. In modern medicine, the clinical symptoms of spleen deficiency are throught to be relevant to the abnormal functions of digestion, absorption and metabolism. To elucidate the pathophysiology of spleen deficiency, scholars have investigated the syndrome of spleen deficiency from the perspectives of digestive system, immune system, material and energy metabolism, et al. But the recent studies can't fully reflect the essence of spleen deficiency due to the systemic and complex nature of Chinese medicine syndromes. In recent years, reseaches on the differentially expressed gene profiles of Chinese medicine syndromes develops based on the genomic technology, which can reflect the biological basis of Chinese medicine syndromes from the overall level. Exploration of differentially expressed genes in Chinese medicine syndromes based on gene microarray provides new thoughts and methods to interpret the essence of Chinese medicine syndromes and have made some progress.
In the previous research project on the differentially expressed gene profile of chronic superficial gastritis patients with spleen deficiency syndrome, investigators of our lab found that genes of ribosomal proteins such as ribosomal protein S20(RPS20) showed down-regulated tendency, which was validated by fluorescence quantitative PCR. Subsequently we carried out researches in vitro to identify the gene functions of RPS20on rats'small intestinal epithelial cells (IEC-6) and the research results preliminarily showed that genes of ribosomal protein may play an important role in the process of the development of spleen deficiency syndrome. After RNA interference (RNAi) for RPS20gene, the morphology and structure of IEC-6cells were changed and the migration and proliferation function of IEC-6cells were both suppressed, which indicated that down-regulated expression of RPS20gene may influence the digestion and absorption function and mucosal repair function of intestinal epithelial cells. Besides, in order to identify the gene functions of RPS20in vivo in the future, investigator selected the effective sequence of RNAi for RPS20gene on mice's colonic tumor cells (CT26), recombinated and constructed the RNAi lentiviral vectors for RPS20.
Based on the previous studies, firstly we aim to carry out further investigations on the gene function of RPS20in this thesis. We will investigate the effects of Chinese medicines that nourish Qi invigorate spleen on the impaired morphology and function of IEC-6after RNAi for RPS20gene, to prove the previous study results from the viewpoint of the medicine effect and to support the findings in the previous researches. Secondly we aim to verify the validity of RNAi sequence for RPS20previously selected on CT26cell and to provide reference for further functional identification of RPS20by RNAi in mice, by producing lentivirus of RNAi for RPS20based on the recombinant lentiviral vector, transfecting CT26cells with it and investigating its effect on the growth of CT26cell.
Methods
1. Transfect IEC-6cells with siRNA and treat them with the extract from Chinese medicines that nourish Qi to invigorate spleen. And then determine proliferation, migration, morphology and structure, RPS20expression and polyamine content of IEC-6cells.
(1) Investigation of the growth conditions of IEC-6cells:Investigate the growth curves of IEC-6cells by the methods of cell counting and xCELLingence real-time cell analyzer (RTCA), to provide reference for choosing the appropriate cell planting density and the right time for interventiont in the following experiments.
(2) Selection of the transfection condition of RNA interference:Add a sequence expressing fluorescence into the RNAi sequence previously selected. Monitor the growth status of IEC-6cells transfected by different transfection mixtures with xCELLingence RTCA, which can reflect the effect of RNA interference. Select the suitable transfection condition according to the growth status and the fluorescence expression of IEC-6cells.
(3) Effect of Chinese medicine on proliferation of IEC-6cells:Use xCELLingence RTCA to study the effects of extracts from Radix Astragali, Radix Glycytthizae and Radix Codonopsis on proliferation of normal IEC-6cells and that of IEC-6cells after RNA interference.
(4) Effect of Chinese medicine on migration of IEC-6cells:Use the migration model of scarification to study the effects of extract from Radix Astragali on migration of normal IEC-6cells and that of IEC-6cells after RNA interference.
(5) Effect of Chinese medicine on morphology and structure of IEC-6cells:Observe the morphology and structure of IEC-6cells after RNA interference and after treatment with extract from Radix Astragali by phase contrast microscope and transmission electron microscope respectively to investigate the restoring effect of Chinese medicine on the impaired morphology and structure of IEC-6cells after RNA interference.
(6) Effect of Chinese medicine on RPS20expression of IEC-6cells:Determine the RPS20mRNA and protein expression of IEC-6cells after RNA interference and after treatment with extract from Radix Astragali by quantitative RT-PCR and Westerblot respectively.
(7) Effect of Chinese medicine on polyamine content of IEC-6cells:Prepare the samples for HPLC determination with IEC-6cells after RNA interference and after treatment with extract from Radix Astragali. And determine the content of spermidine in the samples with the method of HPLC pre-column derivatization.
2. Effect of lentivirus of RNAi for RPS20on the growth of CT26cells:Co-transfect293FT cells with the recombinant lentiviral vectors against RPS20in CT26cells and packing reagents to produce lentivirus of RNAi for RPS20. Determine the titer of lentiviral stocks and transfect CT26with them. Monitor the growth of CT26post-transfection by xCELLigence RTCA to investigate the effect of lentivirus of RNAi on the growth of CT26cells.
Results
1. RNAi for RPS20of IEC-6cells and the effects of Chinese medicines that nourish Qi to invigorate spleen:
(1) The growth conditions of IEC-6cells:The growth curve of IEC-6cells planted with the density of1×104cells/well in the24-well plate was drawn by cell counting, which showed the cells grew into the logarithmic phase from the3rd day after planted and reached the plateau from the10th day. By xCELLigence RTCA, the growth curves of IEC-6cells of different passages (23rd and26th) planted in different densities were obtained. Cells of23rd and26th passages showed similar growth curves in the same planting density. As the planting density increased, the time of which cells'growth reached the plateau shortened.
(2) The transfection condition of RNA interference:The results from RTCA showed the transfection agent (Lipofectamine2000) of high and medium dosage was significantly harmful to the cells. Transfection mixtures of different dose-combinations showed similar inhibitory effects on the growth of IEC-6cells at the beginning of transfection, but the inhibitory effects of mixtures with higher dosage of siRNA lasted longer. Considering the fluorescence expression with the growth status of the cells, we selected the transfection condition as follow:0.3μL Lipofectamine2000and16pmol siRNA per well in the E-plate, the concentrations of which were1.5mg/L and80nmol/L respectively.
(3) Effect of Chinese medicine on proliferation of IEC-6cells:Extracts from Radix Astragali and Radix Codonopsis promoted proliferation of normal IEC-6cells, but the effect of Radix Glycytthizae on normal IEC-6cells was not significant. Proliferation of IEC-6cells after RNA interference was suppressed. Extracts from Radix Astragali, Radix Glycytthizae and Radix Codonopsis significantly promoted proliferation of IEC-6cells after RNA interference. The effect of Radix Astragali was better. The results showed that Chinese medicines that invigorates spleen can restore the pathology of depressed proliferation of IEC-6cells after RNA interference.
(4) Effect of Chinese medicine on migration of IEC-6cells:High and medium dosage of extract from Radix Astragali could effectively promote migration of normal IEC-6cells which were wounded of scarification. Migration of IEC-6cells after RNA interference was suppressed, which was significantly enhanced after treatment with extract from Radix Astragali, compared with the transfected group. But it wasn't restored to the normal level. The results showed that the impaired function of migration of IEC-6cells after RNA interference can be restored to some extent by Chinese medicines that invigorates spleen.
(5) Effect of Chinese medicine on morphology and structure of IEC-6cells:It was observed by optical microscope that ceH numbers of IEC-6cells were decreased, cell morphology was changed and intercellular spaces were enlarged after RNA interference, which was improved after treatment with extract from Radix Astragali. The increasing of endochylema vacuolization and lysosome numbers and the reduction of mitochondria was observed by transmission electron microscope. The impaired ultrastructure of cells was restored to some extent after treatment.
(6) Effect of Chinese medicine on RPS20expression of IEC-6cells:The expressions of RPS20mRNA of IEC-6cells were significantly inhibited after RNA interference, which could be enhanced after treatment with extract from Radix Astragali. The group of medium dosage showed significant difference compared with the group of transfection. The expressions of RPS20protein were also inhibited markedly after RNA interference, but they weren't elevated after treatment.
(7) Effect of Chinese medicine on polyamine content of IEC-6cells:The regression equation of spermidine was Y=25.69X-1.337and its linear correlation coefficient R=0.9998, which showed spermidine was of good linearity in the range from0.7600to7.600nmol. The contents of spermidine of IEC-6cells after RNA interference were increased but without significant difference compared with control group. The contents of spermidine of IEC-6cells in groups treated with extract from Radix Astragali were markedly higher than that of control groups, but without significant difference compared with the transfection group.
2. Effect of lentivirus of RNAi for RPS20on the growth of CT26cells:The titer of lentiviral stocks was1.2X105TU/mL. The increasing of cell index of CT26cells was significantly inhibited after transfected by the lentivirus of RNAi and the inhibitory rate of cell index reached82.24%30hours after transfection. The results showed that the RNAi sequence selected previously can be constructed into lentivirus successfully and inhibit the growth of CT26cells after transfection effectively.
Conelusions
Transfecting IEC-6cells by the RNA interference condition selected based on the results of xCELLigence RTCA determination and cell fluorescence expression can effectively inhibit RPS20expression of IEC-6cell and also inhibit its ability of proliferation and migration and change its morphology and structure. After treatment with extract from Radix Astragali, the expression of RPS20mRNA of IEC-6cell can be enhanced, the ability of proliferation and migration of IEC-6cell can be promoted and the impaired morphology and structure of IEC-6cell can be restored. The result that the abnormal morphology and function of IEC-6cell initiated by deficient expression of RPS20can be improved after treatment with Chinese medicine that nourishes Qi to invigorate spleen, suggests that RPS20may be relevant to the development of spleen deficiency syndrome and further verified the findings of our previous research on the differentially expressed genes of spleen deficiency syndrome and the results of RPS20functional identification of our research team. Our research can provide a reference for functional identification on differentially expressed genes of Chinese medicine syndromes.
Results in this thesis also confirmed that the recombinant lentiviral vectors for RPS20on CT26cells can be used to produce lentiviral stock of RNAi successfully and transfecting CT26cells with it can inhibit cell growth effectively, which suggests that lentivirus of RNAi may be used to identify RPS20function in the further study in mice.
脾虚证是中医辨证的常见证型,现代医学认为脾虚证的临床表现多与消化吸收和代谢功能异常相关,国内外学者从消化系统、免疫系统、物质能量代谢等角度对脾虚证进行了大量的探索研究,初步阐释脾虚证的现代病理生理学基础。由于中医证候具有系统性和复杂性,局部的研究难以全面地反映脾虚证的本质。近年来,基于基因组学技术的中医证候基因差异表达谱研究,可从整体水平反映中医证候的生物学基础,应用基因芯片等技术对中医证候差异表达基因的探讨分析为阐明中医证候的本质提供了新的思路和方法,并取得了一定的进展。
本实验室在前期课题对慢性浅表性胃炎脾虚证患者胃粘膜基因差异表达谱的研究中发现,脾虚证患者核糖体蛋白S20(RPS20)等基因表达出现下调趋势,并通过荧光定量PCR验证了RPS20等基因的表达下调呈阳性结果。后续对RPS20基因的体外功能研究结果初步表明核糖体蛋白基因在脾虚证发生发展过程中可能具有重要作用,RPS20基因RNA干扰后大鼠小肠上皮细胞(IEC-6)的形态结构发生改变,细胞的迁移、增殖能力受到抑制,提示RPS20基因表达下调可能影响小肠上皮细胞的消化吸收和粘膜损伤修复功能。此外通过小鼠结肠腺癌细胞(CT26)筛选了针对小鼠RPS20基因的RNA干扰有效序列,重组构建慢病毒RNA干扰表达载体,以期在动物体内对RPS20基因进行后续的功能研究。
本研究将在前期研究的基础上进一步开展RPS20基因的功能鉴定,通过考察益气健脾中药对PRS20基因RNA干扰后IEC-6细胞受损的形态和功能的作用,从“药物作用”的角度反证前期的研究结果,进一步探讨研究RPS20的基因功能,以支持课题组前期的研究发现。此外,根据前期构建的RNA干扰表达载体包装RNA干扰慢病毒,考察其转染后对CT26细胞生长的影响,进一步验证前期所筛选的RNA干扰序列的有效性,并为后续在小鼠体内采用RNA干扰技术进行RPS20基因功能研究提供技术参考。
研究方法
1.采用siRNA转染IEC-6细胞对RPS20基因进行RNA干扰,给予益气健脾中药提取物作用后,检测IEC-6细胞的增殖和迁移能力、细胞形态结构、RPS20表达情况和细胞内的多胺含量。
(1)IEC-6细胞生长情况的考察：分别采用细胞计数法和xCELLigence实时细胞分析仪(RTCA)考察IEC-6细胞的生长曲线,为后续实验选择合适的细胞接种密度和实验干预时间点提供参考。
(2)选择RNA干扰的转染条件：在前期合成的siRNA干扰序列的基础上加入荧光表达序列,设置不同的转染条件,采用RTCA检测转染后IEC-6细胞的生长情况,以转染后细胞的生长情况直接反映RNA干扰的效果,并结合转染后细胞的荧光表达情况选择合适的转染条件。
(3)健脾中药对IEC-6细胞增殖能力的作用：采用RTCA检测益气健脾中药黄芪、甘草、党参提取物对正正常IEC-6细胞和RNA干扰后IEC-6细胞增殖能力的影响,并选择作用效果较明显的中药提取物进行后续实验研究。
(4)健脾中药对IEC-6细胞迁移能力的作用：采用IEC-6细胞划痕损伤迁移模型,研究黄芪提取物对正正常IEC-6细胞和RNA干扰后IEC-6细胞在划痕损伤后迁移能力的影响。
(5)健脾中药对IEC-6细胞形态结构的作用：分别采用光学显微镜和透射电镜观察RNA干扰和给予黄芪提取物作用后IEC-6细胞形态和结构的变化情况,考察中药对RNA干扰后IEC-6细胞受损的形态结构的恢复作用。
(6)健脾中药对IEC-6细胞RPS20表达水平的作用：分别采用荧光定量PCR和Westernblot检测RNA干扰和给予黄芪提取物作用后IEC-6细胞中RPS20mRNA和蛋白的表达水平。
(7)健脾中药对IEC-6细胞中多胺含量的影响：对RNA干扰和黄芪提取物作用后的IEC-6细胞进行样品处理,采用苯甲酰氯柱前衍生反相高效液相色谱法检测各样品中精脒的含量。
2.RNA干扰慢病毒对CT26细胞生长的影响：将前期重组构建的针对CT26细胞的慢病毒表达载体与慢病毒包装试剂共转染293FT细胞,包装获得RNA干扰慢病毒,测定病毒滴度并将其转染CT26细胞,采用RTCA检测转染后细胞的生长情况,研究RNA干扰慢病毒对CT26细胞生长的影响。
研究结果
1.RNA干扰IEC-6细胞和益气健脾中药的作用：
(1)IEC-6细胞的生长情况：细胞计数法绘制以1×104cells/well的密度接种于24孔培养板中IEC-6细胞的生长曲线,细胞从接种的第3天开始进入对数生长期,接种后第10天进入缓慢生长的平台期；RTCA检测得到不同接种密度的23代和26代IEC-6细胞的生长曲线,在相同接种密度条件下,23代和26代的IEC-6细胞生长趋势相似,随着接种密度的增大,细胞生长达到平台期的时间缩短。
(2)RNA干扰的转染条件：RTCA检测结果发现高剂量和中剂量的转染试剂(Lipofectamine2000)对细胞生长有明显毒害作用；不同剂量组合的转染混合物在转染初期对IEC-6细胞生长的抑制作用相近,但在相同转染试剂用量时siRNA剂量的增加能延长对细胞生长抑制作用的持续时间。结合荧光表达的情况,最后选择RNA干扰的转染条件为：E-Plate16培养板中每孔加入0.3μL的Lipofectamine2000和16pmol的siRNA(浓度分别为1.5mg/L和80nmol/L)。
(3)健脾中药对IEC-6细胞增殖能力的作用：黄芪和党参提取物能促进正正常IEC-6细胞的增殖,甘草对正常IEC-6细胞的作用不明显；RNA干扰后IEC-6细胞的增殖能力下降,而黄芪、甘草和党参提取物都能有效促进RNA干扰后IEC-6细胞的增殖,其中黄芪的效果较优。结果表明健脾中药可在一定程度上逆转RPS20基因干扰引发的IEC-6细胞增殖能力下降的病理表现。
(4)健脾中药对IEC-6细胞迁移能力的作用：高剂量和中剂量的黄芪提取物能有效促进正常IEC-6细胞在划痕损伤后的迁移能力；RNA干扰后IEC-6细胞的迁移能力显著下降,经黄芪提取物作用后其迁移能力较转染模型组显著提高,但未能恢复到正正常水平。结果表明RPS20基因干扰后IEC-6细胞受损的迁移能力可在健脾中药作用后得到一定程度的恢复。
(5)健脾中药对IEC-6细胞形态结构的作用：光学显微镜观察发现RNA干扰后IEC-6细胞数目减少,细胞形态改变,细胞间隙增大,给予黄芪提取物作用后细胞受损的形态得到不同程度的改善；透射电镜观察发现RNA干扰后IEC-6细胞胞浆中空泡和溶酶体数目增多,线粒体减少,经治疗后细胞受损结构得到一定程度的改善。
(6)健脾中药对IEC-6细胞RPS20表达的作用：RNA干扰后IEC-6细胞中RPS20mRNA表达被显著抑制,给予黄芪提取物作用后其表达量有所提高,其中中剂量组与转染模型组比较具有显著差异性；RPS20蛋白表达在RNA干扰后被显著抑制,但经健脾中药作用后RPS20蛋白表达没有提高。
(7)健脾中药对IEC-6细胞中多胺含量的影响：精脒对照品的回归方程为Y=25.69X—1.337,相关系数R=0.9998,精脒对照品在0.7600～7.600nmol范围内线性良好。检测结果RNA干扰后IEC-6细胞中精脒含量比正正常组增加,但无统计学意义；经黄芪提取物作用后的各组细胞中精脒含量较正正常组显著增加,但与RNA干扰模型组比较无明显差异。
2.RNA干扰慢病毒对CT26细胞生长的影响：慢病毒滴度为1.2×105TU/mL,RNA干扰慢病毒转染使CT26细胞指数的增长受到明显抑制,转染后30h细胞指数抑制率达到82.24%。结果表明根据前期筛选的RNA干扰序列包装获得的慢病毒转染后能有效抑制CT26细胞中RPS20的表达从而抑制细胞的生长。
结论
根据xCELLigence RTCA检测结果和细胞荧光表达情况选择的RNA干扰条件转染IEC-6细胞,可有效抑制IEC-6细胞RPS20的表达,并抑制细胞的增殖和迁移能力,影响细胞的形态结构；给予益气健脾中药黄芪的提取物作用后可提高RNA干扰后IEC-6细胞中RPS20mRNA的表达,促进细胞的增殖和迁移,并在一定程度上改善细胞受损的形态结构；给予益气健脾中药作用后,能改善RPS20表达异常所导致的细胞形态和功能的异常,提示RPS20可能与脾虚证的症状表现之间存在一定联系,本研究从药物作用的角度进一步支持课题组前期对脾虚证差异表达基因的研究发现及对RPS20在IEC-6细胞的生物功能鉴定的研究结果；本研究可为中医证候差异表达基因的功能研究提供参考。
本研究还验证了前期针对CT26细胞重组构建的慢病毒表达载体可成功用于包装RNA干扰慢病毒,以其转染CT26细胞能有效抑制细胞的生长,提示RNA干扰慢病毒可能用于小鼠体内RNA干扰进行RPS20功能鉴定的后续研究。
Background and Objective
Spleen deficiency is a common syndrome of Chinese medicine. In modern medicine, the clinical symptoms of spleen deficiency are throught to be relevant to the abnormal functions of digestion, absorption and metabolism. To elucidate the pathophysiology of spleen deficiency, scholars have investigated the syndrome of spleen deficiency from the perspectives of digestive system, immune system, material and energy metabolism, et al. But the recent studies can't fully reflect the essence of spleen deficiency due to the systemic and complex nature of Chinese medicine syndromes. In recent years, reseaches on the differentially expressed gene profiles of Chinese medicine syndromes develops based on the genomic technology, which can reflect the biological basis of Chinese medicine syndromes from the overall level. Exploration of differentially expressed genes in Chinese medicine syndromes based on gene microarray provides new thoughts and methods to interpret the essence of Chinese medicine syndromes and have made some progress.
In the previous research project on the differentially expressed gene profile of chronic superficial gastritis patients with spleen deficiency syndrome, investigators of our lab found that genes of ribosomal proteins such as ribosomal protein S20(RPS20) showed down-regulated tendency, which was validated by fluorescence quantitative PCR. Subsequently we carried out researches in vitro to identify the gene functions of RPS20on rats'small intestinal epithelial cells (IEC-6) and the research results preliminarily showed that genes of ribosomal protein may play an important role in the process of the development of spleen deficiency syndrome. After RNA interference (RNAi) for RPS20gene, the morphology and structure of IEC-6cells were changed and the migration and proliferation function of IEC-6cells were both suppressed, which indicated that down-regulated expression of RPS20gene may influence the digestion and absorption function and mucosal repair function of intestinal epithelial cells. Besides, in order to identify the gene functions of RPS20in vivo in the future, investigator selected the effective sequence of RNAi for RPS20gene on mice's colonic tumor cells (CT26), recombinated and constructed the RNAi lentiviral vectors for RPS20.
Based on the previous studies, firstly we aim to carry out further investigations on the gene function of RPS20in this thesis. We will investigate the effects of Chinese medicines that nourish Qi invigorate spleen on the impaired morphology and function of IEC-6after RNAi for RPS20gene, to prove the previous study results from the viewpoint of the medicine effect and to support the findings in the previous researches. Secondly we aim to verify the validity of RNAi sequence for RPS20previously selected on CT26cell and to provide reference for further functional identification of RPS20by RNAi in mice, by producing lentivirus of RNAi for RPS20based on the recombinant lentiviral vector, transfecting CT26cells with it and investigating its effect on the growth of CT26cell.
Methods
1. Transfect IEC-6cells with siRNA and treat them with the extract from Chinese medicines that nourish Qi to invigorate spleen. And then determine proliferation, migration, morphology and structure, RPS20expression and polyamine content of IEC-6cells.
(1) Investigation of the growth conditions of IEC-6cells:Investigate the growth curves of IEC-6cells by the methods of cell counting and xCELLingence real-time cell analyzer (RTCA), to provide reference for choosing the appropriate cell planting density and the right time for interventiont in the following experiments.
(2) Selection of the transfection condition of RNA interference:Add a sequence expressing fluorescence into the RNAi sequence previously selected. Monitor the growth status of IEC-6cells transfected by different transfection mixtures with xCELLingence RTCA, which can reflect the effect of RNA interference. Select the suitable transfection condition according to the growth status and the fluorescence expression of IEC-6cells.
(3) Effect of Chinese medicine on proliferation of IEC-6cells:Use xCELLingence RTCA to study the effects of extracts from Radix Astragali, Radix Glycytthizae and Radix Codonopsis on proliferation of normal IEC-6cells and that of IEC-6cells after RNA interference.
(4) Effect of Chinese medicine on migration of IEC-6cells:Use the migration model of scarification to study the effects of extract from Radix Astragali on migration of normal IEC-6cells and that of IEC-6cells after RNA interference.
(5) Effect of Chinese medicine on morphology and structure of IEC-6cells:Observe the morphology and structure of IEC-6cells after RNA interference and after treatment with extract from Radix Astragali by phase contrast microscope and transmission electron microscope respectively to investigate the restoring effect of Chinese medicine on the impaired morphology and structure of IEC-6cells after RNA interference.
(6) Effect of Chinese medicine on RPS20expression of IEC-6cells:Determine the RPS20mRNA and protein expression of IEC-6cells after RNA interference and after treatment with extract from Radix Astragali by quantitative RT-PCR and Westerblot respectively.
(7) Effect of Chinese medicine on polyamine content of IEC-6cells:Prepare the samples for HPLC determination with IEC-6cells after RNA interference and after treatment with extract from Radix Astragali. And determine the content of spermidine in the samples with the method of HPLC pre-column derivatization.
2. Effect of lentivirus of RNAi for RPS20on the growth of CT26cells:Co-transfect293FT cells with the recombinant lentiviral vectors against RPS20in CT26cells and packing reagents to produce lentivirus of RNAi for RPS20. Determine the titer of lentiviral stocks and transfect CT26with them. Monitor the growth of CT26post-transfection by xCELLigence RTCA to investigate the effect of lentivirus of RNAi on the growth of CT26cells.
Results
1. RNAi for RPS20of IEC-6cells and the effects of Chinese medicines that nourish Qi to invigorate spleen:
(1) The growth conditions of IEC-6cells:The growth curve of IEC-6cells planted with the density of1×104cells/well in the24-well plate was drawn by cell counting, which showed the cells grew into the logarithmic phase from the3rd day after planted and reached the plateau from the10th day. By xCELLigence RTCA, the growth curves of IEC-6cells of different passages (23rd and26th) planted in different densities were obtained. Cells of23rd and26th passages showed similar growth curves in the same planting density. As the planting density increased, the time of which cells'growth reached the plateau shortened.
(2) The transfection condition of RNA interference:The results from RTCA showed the transfection agent (Lipofectamine2000) of high and medium dosage was significantly harmful to the cells. Transfection mixtures of different dose-combinations showed similar inhibitory effects on the growth of IEC-6cells at the beginning of transfection, but the inhibitory effects of mixtures with higher dosage of siRNA lasted longer. Considering the fluorescence expression with the growth status of the cells, we selected the transfection condition as follow:0.3μL Lipofectamine2000and16pmol siRNA per well in the E-plate, the concentrations of which were1.5mg/L and80nmol/L respectively.
(3) Effect of Chinese medicine on proliferation of IEC-6cells:Extracts from Radix Astragali and Radix Codonopsis promoted proliferation of normal IEC-6cells, but the effect of Radix Glycytthizae on normal IEC-6cells was not significant. Proliferation of IEC-6cells after RNA interference was suppressed. Extracts from Radix Astragali, Radix Glycytthizae and Radix Codonopsis significantly promoted proliferation of IEC-6cells after RNA interference. The effect of Radix Astragali was better. The results showed that Chinese medicines that invigorates spleen can restore the pathology of depressed proliferation of IEC-6cells after RNA interference.
(4) Effect of Chinese medicine on migration of IEC-6cells:High and medium dosage of extract from Radix Astragali could effectively promote migration of normal IEC-6cells which were wounded of scarification. Migration of IEC-6cells after RNA interference was suppressed, which was significantly enhanced after treatment with extract from Radix Astragali, compared with the transfected group. But it wasn't restored to the normal level. The results showed that the impaired function of migration of IEC-6cells after RNA interference can be restored to some extent by Chinese medicines that invigorates spleen.
(5) Effect of Chinese medicine on morphology and structure of IEC-6cells:It was observed by optical microscope that ceH numbers of IEC-6cells were decreased, cell morphology was changed and intercellular spaces were enlarged after RNA interference, which was improved after treatment with extract from Radix Astragali. The increasing of endochylema vacuolization and lysosome numbers and the reduction of mitochondria was observed by transmission electron microscope. The impaired ultrastructure of cells was restored to some extent after treatment.
(6) Effect of Chinese medicine on RPS20expression of IEC-6cells:The expressions of RPS20mRNA of IEC-6cells were significantly inhibited after RNA interference, which could be enhanced after treatment with extract from Radix Astragali. The group of medium dosage showed significant difference compared with the group of transfection. The expressions of RPS20protein were also inhibited markedly after RNA interference, but they weren't elevated after treatment.
(7) Effect of Chinese medicine on polyamine content of IEC-6cells:The regression equation of spermidine was Y=25.69X-1.337and its linear correlation coefficient R=0.9998, which showed spermidine was of good linearity in the range from0.7600to7.600nmol. The contents of spermidine of IEC-6cells after RNA interference were increased but without significant difference compared with control group. The contents of spermidine of IEC-6cells in groups treated with extract from Radix Astragali were markedly higher than that of control groups, but without significant difference compared with the transfection group.
2. Effect of lentivirus of RNAi for RPS20on the growth of CT26cells:The titer of lentiviral stocks was1.2X105TU/mL. The increasing of cell index of CT26cells was significantly inhibited after transfected by the lentivirus of RNAi and the inhibitory rate of cell index reached82.24%30hours after transfection. The results showed that the RNAi sequence selected previously can be constructed into lentivirus successfully and inhibit the growth of CT26cells after transfection effectively.
Conelusions
Transfecting IEC-6cells by the RNA interference condition selected based on the results of xCELLigence RTCA determination and cell fluorescence expression can effectively inhibit RPS20expression of IEC-6cell and also inhibit its ability of proliferation and migration and change its morphology and structure. After treatment with extract from Radix Astragali, the expression of RPS20mRNA of IEC-6cell can be enhanced, the ability of proliferation and migration of IEC-6cell can be promoted and the impaired morphology and structure of IEC-6cell can be restored. The result that the abnormal morphology and function of IEC-6cell initiated by deficient expression of RPS20can be improved after treatment with Chinese medicine that nourishes Qi to invigorate spleen, suggests that RPS20may be relevant to the development of spleen deficiency syndrome and further verified the findings of our previous research on the differentially expressed genes of spleen deficiency syndrome and the results of RPS20functional identification of our research team. Our research can provide a reference for functional identification on differentially expressed genes of Chinese medicine syndromes.
Results in this thesis also confirmed that the recombinant lentiviral vectors for RPS20on CT26cells can be used to produce lentiviral stock of RNAi successfully and transfecting CT26cells with it can inhibit cell growth effectively, which suggests that lentivirus of RNAi may be used to identify RPS20function in the further study in mice.
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