三氧化二砷对急性哮喘小鼠CD4~+T细胞凋亡的影响及机制研究
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摘要
第一章三氧化二砷对急性哮喘小鼠气道高反应性及气道炎症的影响
目的
(1)建立急性哮喘小鼠模型,并鉴定建立的模型是否成功；
(2)观察三氧化二砷对急性哮喘小鼠气道高反应性及气道炎症的影响。
方法
将30只SPF级BALB/c雌性小鼠随机分为三组：正常组(PBS组)、哮喘组(OVA/PBS组)和三氧化二砷组(OVA/ATO组),每组10只。所有小鼠适应性饲养一周后按以下方法建立急性哮喘小鼠模型。PBS组于第1、13天经腹腔注射PBS缓冲液(PH7.2-7.4)0.2ml,第19-24天连续6天每天予以6ml PBS缓冲液(PH7.2-7.4)雾化30min,每次雾化前30min予以腹腔注射PBS缓冲液(PH7.2-7.4)0.2ml; OVA/PBS组于第1、13天经腹腔注射OVA与氢氧化铝凝胶的混合溶液(10μgOVA(GradeV)和2mg氢氧化铝凝胶溶于PBS缓冲液中,充分混匀)0.2ml,第19-24天连续6天每天予以5%OVA (GradeV)溶液6ml雾化30min,每次雾化前30mmin予以腹腔注射PBS缓冲液(PH7.2-7.4)0.2ml; OVA/ATO组于第1、13天腹腔注射OVA与氢氧化铝凝胶的混合溶液(10μg OVA(GradeV)和2mg氢氧化铝凝胶溶于PBS缓冲液中,充分混匀)0.2ml,第19-24天连续6天每天予以5%OVA (GradeV)溶液6ml雾化30min,每次雾化前30mmin予以腹腔注射三氧化二砷溶液(用量：2.5mg/kgx小鼠体重,配成0.2ml溶液)。第25天处理所有小鼠,检测以下指标：(1)对小鼠的一般行为活动进行观察；(2)不同浓度的乙酰甲胆碱激发小鼠后,采用有创肺阻抗法测定小鼠的气道反应性；(3)收集BALF行细胞计数及分类；(4)肺组织病理切片观察炎症细胞浸润及黏液分泌情况。计量资料以均数±标准差(x±S)表示。各组数据先行正态检验和方差齐性检验,如果数据不符合正态分布,则经自然对数转换为正态分布的数据。若数据方差不齐,则行Dunnett's T3法。多组均数间比较采用单因素方差分析(Analysis of Variance, ANOVA),各组间两两比较采用LSD检验。P<0.05表示差异有统计学意义。
结果
实验前：各组小鼠的外观、行为、对刺激的反应、活动及体重均无明显差异。致敏阶段：各组小鼠的腹腔注射部位无红肿、溃烂。激发阶段：PBS组小鼠饮食正常,活动灵敏,皮毛清洁有光泽,OVA/PBS组小鼠早期出现兴奋、打喷嚏、抓耳挠腮,后期出现匍匐不起、嗜睡症状,OVA/ATO组小鼠出现打喷嚏、兴奋的症状较OVA/PBS组轻,无嗜睡症状。而且,实验全程OVA/ATO组小鼠没有活动减少、厌食、体重减轻等异常症状。
OVA/PBS组小鼠气道反应性较PBS组显著增高(P<0.05)；而OVA/ATO组小鼠气道反应性较OVA/PBS组明显降低(P<0.05)。
OVA/PBS组小鼠BALF中白细胞总数为21.11±3.28×104/ml、嗜酸性粒细胞数为3.10±0.50×104/ml、淋巴细胞数为6.60±0.97×104/ml、中性粒细胞数为4.92±0.45×104/ml,较PBS组均显著增加(均P<0.01)；OVA/ATO组BALF中白细胞总数为13.04±2.58×104/ml、嗜酸性粒细胞数为1.06±0.19×104/ml、淋巴细胞数为2.43±0.28×104/ml、中性粒细胞数为2.36±0.29×104/ml,较OVA/PBS组均明显降低(均P<0.01)。
与PBS组相比,OVA/PBS组小鼠肺组织大量炎症细胞浸润、杯状细胞增生、气道分泌大量黏液(P<0.01); OVA/ATO组气道炎症和黏液分泌状态较OVA/PBS组有所减轻(P<0.05)。
结论
(1)本实验中建立的急性哮喘小鼠模型是成功的；
(2)三氧化二砷能减轻急性哮喘小鼠的气道高反应性及气道炎症。
第二章三氧化二砷诱导急性哮喘小鼠CD4+T细胞凋亡
目的
检测三氧化二砷干预对急性哮喘小鼠CD4+T细胞凋亡的影响。
方法
在体部分：将18只SPF级BALB/c雌性小鼠随机分为三组：正常组(PBS组)、哮喘组(OVA/PBS组)和三氧化二砷组(OVA/ATO组),每组6只,按第一章中的方法建立急性哮喘小鼠模型,第25天处理小鼠,利用免疫磁珠分离纯化各组小鼠脾CD4+T细胞,计数CD4+T细胞总数,然后用完全性RPMI-1640培养基按2×106cell/ml的浓度接种细胞,同时加入ConA (5ug/ml)进行刺激,置于培养箱中培养24h,用流式细胞术检测细胞的凋亡率。
离体部分：免疫磁珠分离纯化OVA/PBS组小鼠脾CD4+T细胞,然后用完全性RPMI-1640培养基按2×106cell/ml的浓度接种细胞,加入ConA (5ug/ml)进行刺激,同时分别加入四组不同浓度的三氧化二砷溶液(0μM,1μM,3μM,5μM)培养20小时,用流式细胞术检测细胞的凋亡率。计量资料以均数±标准差(x±S)表示。各组数据先行正态检验和方差齐性检验,如果数据不符合正态分布,则经自然对数转换为正态分布的数据。若数据方差不齐,则行Dunnett's T3法。多组均数间比较采用单因素方差分析(Analysis of Variance, ANOVA),各组间两两比较采用LSD检验。P<0.05表示差异有统计学意义。
结果
在体部分：OVA/PBS组小鼠脾CD4+T细胞总数为(17.88±2.13)×108/L,较PBS组显著增多(P<0.01)；OVA/ATO组小鼠脾CD4+T细胞总数为(12.92±2.31)×108/L,较OVA/PBS组显著减少(P<0.05)。OVA/PBS组CD4+T细胞的凋亡率为20.69±2.68%,较PBS组显著下降(P<0.05)；OVA/ATO组CD4+T细胞的凋亡率为34.71±0.98%,较OVA/PBS组显著升高(P<0.05)。
离体部分：3μM组CD4+T细胞的凋亡率为31.85±3.73%,较0μM组显著升高(P<0.05)；5μM组CD4+T细胞的凋亡率为40.54±1.99%,较0μM组和3μM组均显著升高(P<0.01和P<0.05)。
结论
三氧化二砷诱导急性哮喘小鼠CD4+T细胞的凋亡。
第三章内质网应激-CHOP途径参与三氧化二砷诱导CD4+T细胞的凋亡
目的
(1)探讨三氧化二砷干预对CD4+T细胞GRP78和CHOP蛋白表达的影响；
(2)探索CHOP蛋白在三氧化二砷诱导CD4+T细胞凋亡中的作用。
方法
第一部分：免疫磁珠分离纯化第一章中已建模成功的OVA/PBS组小鼠脾CD4+T细胞,体外加入5μM三氧化二砷,分别培养0h,2.5h,5h,7.5h后,检测蛋白GRP78. CHOP的表达。
第二部分：将OVA/PBS组小鼠脾CD4+T细胞分成两组：CHOP siRNA组和control siRNA组。首先,用可以沉默CHOP表达的CHOP siRNA和无沉默效果的对照片段contol siRNA分别转染两组CD4+T细胞,转染成功后,用real-timePCR和western blot分别检测两组CHOP mRNA和蛋白的表达,评估siRNA的沉默效果,达到满意效果后加入5μM三氧化二砷培养20h,检测各组细胞的凋亡率。
计量资料以均数±标准差(x±S)表示。各组数据先行正态检验和方差齐性检验,如果数据不符合正态分布,则经自然对数转换为正态分布的数据。若数据方差不齐,则行Dunnett's T3法。两组均数间比较采用独立样本t检验,多组均数间比较采用单因素方差分析(Analysis of Variance, ANOVA),各组间两两比较采用LSD检验。P<0.05表示差异有统计学意义。
结果
ATO干预2.5h组,5h组,7.5h组GRP78蛋白的相对表达量较0h组均显著增多(分别是P<0.05,P<0.01,P<0.05)；5h组GRP78蛋白的相对表达量较2.5h组和7.5h组均显著升高(均P<0.05)；而2.5h组GRP78蛋白的相对表达量与7.5h组之间未见明显组间差异(P>0.05)。
ATO干预5h组CHOP蛋白的相对表达量较0h组、2.5h组和7.5h组均显著增多(均P<0.05)；
CHOP siRNA组CHOP mRNA和蛋白的表达量较control siRNA组均显著下降(均P<0.01)；
予以ATO干预后CHOP siRNA组CD4+T细胞的凋亡率为32.39±2.30%,较control siRNA组显著下降(P<0.05)。
结论
(1)三氧化二砷能影响CD4+T细胞中GRP78蛋白和CHOP蛋白的表达；
(2)转染CHOP siRNA能沉默CHOP蛋白的表达,并能部分阻断三氧化二砷诱导的CD4+T细胞的凋亡；
(3)内质网应激-CHOP途径参与三氧化二砷诱导的CD4+T细胞的凋亡。
Chapter1The effect of Arsenic trioxide on airway hyperreactivity and airway inflamation in the acute asthma mouse
Objective
(1Establishing a mouse model of acute asthma, and identify the model;
(2)Observe the effect of ATO (Arsenic Trioxide, ATO) on airway hyperreactivity and airway inflamation in the acute asthma mouse.
Method
Thirty SPF BALB/c female mice were randomly divided into three groups:the normal group(PBS group), the asthma group(OVA/PBS group) and the ATO group(OVA/ATO group), each group contains10mice. Establishing a mouse model of acute asthma after all the mice are raise adaptively for one week according to the following methods. The mice of PBS group were intraperitoneal injected with0.2ml PBS buffer(PH7.2-7.4) at the1st and13th day, and were atomized with6ml PBS buffer(PH7.2-7.4) for30min from the19th to the24th day successively (sum6days). The mice of the PBS group received the intraperitoneal injection of0.2ml PBS buffer(PH7.2-7.4)30min before each atomization. The mice of OVA/PBS group were intraperitoneal injected with0.2ml mixed solution of OVA(ovalbumin, OVA) and aluminium hydroxide gel(10μg OVA and2mg aluminium hydroxide gel were dissolved in PBS buffer thoroughly) at the1st and13th day, and were atomized with6ml5%OVA(Grade V) for30min from the19th to the24th day successively (sum6days). The mice of the OVA/PBS group received the intraperitoneal injection of0.2ml PBS buffer(PH7.2-7.4)30min before each atomization. The mice of OVA/ATO group were intraperitoneal injected with0.2ml mixed solution of OVA and aluminium hydroxide gel(10μg OVA and2mg aluminium hydroxide gel were dissolved in PBS buffer thoroughly) at the1st and13th day, and were atomized with6ml 5%OVA(Grade V) for30min from the19th to the24th day successively (sum6days). The mice of the OVA/ATO group received the intraperitoneal injection of0.2ml ATO solution (2.5mg/kgxmouse weights)30min before each atomization. The mice were sacrificed at25th day, the following characters were detected,(1)the ordinary behavior of the mice,(2)airway hyperreactivity was determined after methacholine challenge by invasive pulmonary impedance method,(3) collecting the BALF and carrying out the cell count and cell classification,(4) making lung pathological slices and observing inflammatory cell infiltration and mucus secretion. All the data was presented with Mean±SD, at first, the data was taken test of normality and homogeneity test of variance. If the data does not conform to normal distribution, the natural logarithmic transformation was conducted. In the case of unequal variances, the Dunnett's T3method was conducted. Analysis of Variance(ANOVA) was conducted for Comparison the difference between multiple sets of mean, and the LSD test was conducted for the pairwise comparison of multiple group data. P<0.05brings the statistics significant.
Result
Prior to the experiment, there is no difference between each group mice in appearance, behavior, the response to stimulation, activity and weight. During the sensitization phase, there is no red, swollen and ulcerate in the injection site of each mice. During the challenge phase, the mice of PBS group feed normally and possess sensitive reflection with clean and smooth fur. The mice of OVA/PBS group displayed exciting, sneezing and scratching in early stage, which displayed crawling and somnolence in the later stage. The mice of OVA/ATO group showed mild symptoms of exciting and sneezing than the OVA/PBS group, and didn't show obvious somnolence. Meanwhile, there was no activity decrease, anorexia and weight decrease occurred in the mice of OVA/ATO group.
Compared with the PBS group, airway responsiveness statistically increased in the OVA/PBS group (P<0.05); airway hyperreactivity (AHR) was eased in the OVA/ATO group compared with that in the OVA/PBS group (P<0.05).
In the BALF of OVA/PBS group, the amount of different cell is list as following, the total of leukocytes is21.11±3.28x104/ml, the Eos is3.10±0.50×104/ml, the Lym is6.60±0.97×104/ml, the Neu is4.92±0.45×104/ml, which are significantly increased (all P<0.01) compared with PBS group. In the OVA/ATO group, the amount of different cell is list as following, the total of leukocytes is13.04±2.58×104/ml, the Eos is1.06±0.19×104/ml, the Lym is2.43±0.28×104/ml, the Neu is2.36±0.29×104/ml, which are significantly decreased (all P<0.01) compared with the OVA/PBS group.
Compared with the PBS group, inflammatory cell infiltration, goblet cell hyperplasia and mucus secretion in lung were far more obvious in OVA/PBS group (P<0.01); airway inflammation and mucus hypersecretion of OVA/ATO group were significantly alleviated than OVA/PBS group (P<0.05).
Conclusion
(1)The mouse model of acute asthma established in this experiment was successful.
(2)Treatment of ATO can alleviate the AHR and airway inflammation of the acute asthma mouse.
Chapter2Arsenic Trioxide promotes the CD4+T cells apoptosis in the acute asthma mouse
Objective
Detecting of the effect of arsenic trioxide on the CD4+T cells apoptosis in the acute asthma mouse.
Method
In vivo:Eighteen SPF BALB/c female mice were randomly divided into three groups:the normal group(PBS group), the asthma group(OVA/PBS group) and the ATO group(OVA/ATO group), each group contains6mice. Establishing a mouse model of acute asthma according the method in the chapter1. The mice were sacrificed at25th day, Splenic CD4+T cells of each group were separated and purified by magnetic cell sorting device (MACS). The number of CD4+T cells of each group was counted. Then CD4+T cells were cultured with RPMI-1640according to the concentration of2×106cell/ml, meanwhile, the ConA(5ug/ml) were added. The CD4+T cells were cultured in the incubator(5%C02,37℃)for24hours. When finishing the culture, flow cytometry(FC) was conducted to detect the apoptosis rate of CD4+T cells.
In vitro:Splenic CD4+T cells of mice in the OVA/PBS group were separated and purified by MACS. Then CD4+T cells were cultured with RPMI-1640according to the concentration of2×106cell/ml, meanwhile, the ConA(5ug/ml) and ATO were added. The CD4+T cells were divided into four groups according to the concentration of the added ATO which including OμM group, luM group,3μM group, and5μM group. All the CD4+T cells were cultured in the incubator(5%CO2,37℃) for20hours. When finishing the culture, flow cytometry(FC) was conducted to detect the apoptosis rate of CD4+T cells. All the data was presented with Mean±SD. At first, the data of each group was taken test of normality and homogeneity test of variance. If the data does not conform to normal distribution, the natural logarithmic transformation was conducted. In the case of unequal variances, the Dunnett's T3method was conducted. Analysis of Variance(ANOVA) was conducted for Comparison the difference between multiple sets of mean, and the LSD test was conducted for the pairwise comparison of multiple group data. P<0.05brings the statistics significant.
Result
In vivo, in the OVA/PBS group, the number of spleen CD4+T cells is (17.88±2.13)×108/L, which is significantly increased when compared with PBS group(P<0.01). The number of the OVA/ATO group is (12.92±2.31)×108/L, which is significantly decreased when compared with OVA/PBS group(P<0.05). In OVA/PBS group, the apoptotic rate of CD4+T cells is20.69±2.68%, which is significantly decreased when compared with PBS group(P<0.05). In OVA/ATO group, the apoptotic rate of CD4+T cells is34.71±0.98%, which is significantly increased when compared with OVA/PBS group(P<0.05).
In vitro, in the3μM group, the apoptotic rate of CD4+T cells is31.85±3.73%, which is significantly increased when compared with OμM group(P<0.05). In the5μM group, the apoptotic rate of CD4+T cells is40.54±1.99%, which is significantly increased when compared with OμM group or3μM group (P<0.01or P<0.05).
Conclusion
Arsenic trioxide promotes the CD4+T cell apoptosis in the acute asthma mouse.
Chapter3Endoplasmic reticulum stress-CHOP pathway involved in the CD4+T cell apoptosis which induced by arsenic trioxide
Objective
(1)Probing the effect of arsenic trioxide on the protein expression of GRP78and CHOP in CD4+T cells.
(2)Expounding the role of CHOP protein in the CD4+T cell apoptosis which induced by arsenic trioxide.
Method
Part one, Splenic CD4+T cells of OVA/PBS group were separated and purified by MACS. The CD4+T cells were cultured with5μM ATO for different time which including Oh,2.5h,5h and7.5h. And the protein expression of GRP78and CHOP were detected.
Part two, Splenic CD4+T cells of OVA/PBS group were divided into CHOP siRNA group and control siRNA group. Transfection of CHOP siRNA can silent the protein expression of CHOP. But the control siRNA doesn't have any silent effection which was used as a negative control. First we transfected the CD4+T cells in CHOP siRNA group with CHOP siRNA and transfected the CD4+T cells in control siRNA group with control siRNA. Then we detected the expression of CHOP mRNA by real-time PCR and the protein expression by western blot to estimate the silent effect of CHOP siRNA. After the successful transfection, the CD4+T cells of both group were cultured with5μM ATO for20hours. Flow cytometry(FC) were conducted to detect the apoptotic rate of CD4+T cells.
All the data was presented with Mean±SD. At first, the data of each group was taken test of normality and homogeneity test of variance. If the data does not conform to normal distribution, the natural logarithmic transformation was conducted. In the case of unequal variances, the Dunnett's T3method was conducted. Independent-samples T test were conducted to compare the difference between two groups. Analysis of Variance(ANOVA) was conducted for comparing the difference between multiple sets of mean, and the LSD test was conducted for the pairwise comparison of multiple group data. P<0.05brings the statistics significant.
Result
The protein expression of GRP78in2.5h group,5h group and7.5h group was significantly increased when compared with Oh group (P<0.05, P<0.01, P<0.05respectively). The protein expression of GRP78in5h group also is more than the2.5h group and7.5h group(both P<0.05). There is no obvious difference between2.5h group and7.5h group(P>0.05).
The protein expression of CHOP in5h group is more than Oh group,2.5h group and7.5h group (all P<0.05).
The mRNA and protein expression of CHOP in CHOP siRNA group both significantly decreased when compared with control siRNA group (both P<0.01).
After cultured with ATO, the CD4+T cell apoptotic rate in CHOP siRNA group is32.39±2.30%, which is significantly decreased when compared with control siRNA group (P<0.05).
Conclusion
(1)Arsenic trioxide effects the protein expression of GRP78and CHOP in CD4+T cells.
(2)Transfection of CHOP siRNA silents the protein expression of CHOP and partially blocks the CD4+T cells apoptosis, which induced by arsenic trioxide.
(3)Endoplasmic reticulum stress-CHOP pathway involved in the CD4+T cell apoptosis which induced by arsenic trioxide.
目的
(1)建立急性哮喘小鼠模型,并鉴定建立的模型是否成功；
(2)观察三氧化二砷对急性哮喘小鼠气道高反应性及气道炎症的影响。
方法
将30只SPF级BALB/c雌性小鼠随机分为三组：正常组(PBS组)、哮喘组(OVA/PBS组)和三氧化二砷组(OVA/ATO组),每组10只。所有小鼠适应性饲养一周后按以下方法建立急性哮喘小鼠模型。PBS组于第1、13天经腹腔注射PBS缓冲液(PH7.2-7.4)0.2ml,第19-24天连续6天每天予以6ml PBS缓冲液(PH7.2-7.4)雾化30min,每次雾化前30min予以腹腔注射PBS缓冲液(PH7.2-7.4)0.2ml; OVA/PBS组于第1、13天经腹腔注射OVA与氢氧化铝凝胶的混合溶液(10μgOVA(GradeV)和2mg氢氧化铝凝胶溶于PBS缓冲液中,充分混匀)0.2ml,第19-24天连续6天每天予以5%OVA (GradeV)溶液6ml雾化30min,每次雾化前30mmin予以腹腔注射PBS缓冲液(PH7.2-7.4)0.2ml; OVA/ATO组于第1、13天腹腔注射OVA与氢氧化铝凝胶的混合溶液(10μg OVA(GradeV)和2mg氢氧化铝凝胶溶于PBS缓冲液中,充分混匀)0.2ml,第19-24天连续6天每天予以5%OVA (GradeV)溶液6ml雾化30min,每次雾化前30mmin予以腹腔注射三氧化二砷溶液(用量：2.5mg/kgx小鼠体重,配成0.2ml溶液)。第25天处理所有小鼠,检测以下指标：(1)对小鼠的一般行为活动进行观察；(2)不同浓度的乙酰甲胆碱激发小鼠后,采用有创肺阻抗法测定小鼠的气道反应性；(3)收集BALF行细胞计数及分类；(4)肺组织病理切片观察炎症细胞浸润及黏液分泌情况。计量资料以均数±标准差(x±S)表示。各组数据先行正态检验和方差齐性检验,如果数据不符合正态分布,则经自然对数转换为正态分布的数据。若数据方差不齐,则行Dunnett's T3法。多组均数间比较采用单因素方差分析(Analysis of Variance, ANOVA),各组间两两比较采用LSD检验。P<0.05表示差异有统计学意义。
结果
实验前：各组小鼠的外观、行为、对刺激的反应、活动及体重均无明显差异。致敏阶段：各组小鼠的腹腔注射部位无红肿、溃烂。激发阶段：PBS组小鼠饮食正常,活动灵敏,皮毛清洁有光泽,OVA/PBS组小鼠早期出现兴奋、打喷嚏、抓耳挠腮,后期出现匍匐不起、嗜睡症状,OVA/ATO组小鼠出现打喷嚏、兴奋的症状较OVA/PBS组轻,无嗜睡症状。而且,实验全程OVA/ATO组小鼠没有活动减少、厌食、体重减轻等异常症状。
OVA/PBS组小鼠气道反应性较PBS组显著增高(P<0.05)；而OVA/ATO组小鼠气道反应性较OVA/PBS组明显降低(P<0.05)。
OVA/PBS组小鼠BALF中白细胞总数为21.11±3.28×104/ml、嗜酸性粒细胞数为3.10±0.50×104/ml、淋巴细胞数为6.60±0.97×104/ml、中性粒细胞数为4.92±0.45×104/ml,较PBS组均显著增加(均P<0.01)；OVA/ATO组BALF中白细胞总数为13.04±2.58×104/ml、嗜酸性粒细胞数为1.06±0.19×104/ml、淋巴细胞数为2.43±0.28×104/ml、中性粒细胞数为2.36±0.29×104/ml,较OVA/PBS组均明显降低(均P<0.01)。
与PBS组相比,OVA/PBS组小鼠肺组织大量炎症细胞浸润、杯状细胞增生、气道分泌大量黏液(P<0.01); OVA/ATO组气道炎症和黏液分泌状态较OVA/PBS组有所减轻(P<0.05)。
结论
(1)本实验中建立的急性哮喘小鼠模型是成功的；
(2)三氧化二砷能减轻急性哮喘小鼠的气道高反应性及气道炎症。
第二章三氧化二砷诱导急性哮喘小鼠CD4+T细胞凋亡
目的
检测三氧化二砷干预对急性哮喘小鼠CD4+T细胞凋亡的影响。
方法
在体部分：将18只SPF级BALB/c雌性小鼠随机分为三组：正常组(PBS组)、哮喘组(OVA/PBS组)和三氧化二砷组(OVA/ATO组),每组6只,按第一章中的方法建立急性哮喘小鼠模型,第25天处理小鼠,利用免疫磁珠分离纯化各组小鼠脾CD4+T细胞,计数CD4+T细胞总数,然后用完全性RPMI-1640培养基按2×106cell/ml的浓度接种细胞,同时加入ConA (5ug/ml)进行刺激,置于培养箱中培养24h,用流式细胞术检测细胞的凋亡率。
离体部分：免疫磁珠分离纯化OVA/PBS组小鼠脾CD4+T细胞,然后用完全性RPMI-1640培养基按2×106cell/ml的浓度接种细胞,加入ConA (5ug/ml)进行刺激,同时分别加入四组不同浓度的三氧化二砷溶液(0μM,1μM,3μM,5μM)培养20小时,用流式细胞术检测细胞的凋亡率。计量资料以均数±标准差(x±S)表示。各组数据先行正态检验和方差齐性检验,如果数据不符合正态分布,则经自然对数转换为正态分布的数据。若数据方差不齐,则行Dunnett's T3法。多组均数间比较采用单因素方差分析(Analysis of Variance, ANOVA),各组间两两比较采用LSD检验。P<0.05表示差异有统计学意义。
结果
在体部分：OVA/PBS组小鼠脾CD4+T细胞总数为(17.88±2.13)×108/L,较PBS组显著增多(P<0.01)；OVA/ATO组小鼠脾CD4+T细胞总数为(12.92±2.31)×108/L,较OVA/PBS组显著减少(P<0.05)。OVA/PBS组CD4+T细胞的凋亡率为20.69±2.68%,较PBS组显著下降(P<0.05)；OVA/ATO组CD4+T细胞的凋亡率为34.71±0.98%,较OVA/PBS组显著升高(P<0.05)。
离体部分：3μM组CD4+T细胞的凋亡率为31.85±3.73%,较0μM组显著升高(P<0.05)；5μM组CD4+T细胞的凋亡率为40.54±1.99%,较0μM组和3μM组均显著升高(P<0.01和P<0.05)。
结论
三氧化二砷诱导急性哮喘小鼠CD4+T细胞的凋亡。
第三章内质网应激-CHOP途径参与三氧化二砷诱导CD4+T细胞的凋亡
目的
(1)探讨三氧化二砷干预对CD4+T细胞GRP78和CHOP蛋白表达的影响；
(2)探索CHOP蛋白在三氧化二砷诱导CD4+T细胞凋亡中的作用。
方法
第一部分：免疫磁珠分离纯化第一章中已建模成功的OVA/PBS组小鼠脾CD4+T细胞,体外加入5μM三氧化二砷,分别培养0h,2.5h,5h,7.5h后,检测蛋白GRP78. CHOP的表达。
第二部分：将OVA/PBS组小鼠脾CD4+T细胞分成两组：CHOP siRNA组和control siRNA组。首先,用可以沉默CHOP表达的CHOP siRNA和无沉默效果的对照片段contol siRNA分别转染两组CD4+T细胞,转染成功后,用real-timePCR和western blot分别检测两组CHOP mRNA和蛋白的表达,评估siRNA的沉默效果,达到满意效果后加入5μM三氧化二砷培养20h,检测各组细胞的凋亡率。
计量资料以均数±标准差(x±S)表示。各组数据先行正态检验和方差齐性检验,如果数据不符合正态分布,则经自然对数转换为正态分布的数据。若数据方差不齐,则行Dunnett's T3法。两组均数间比较采用独立样本t检验,多组均数间比较采用单因素方差分析(Analysis of Variance, ANOVA),各组间两两比较采用LSD检验。P<0.05表示差异有统计学意义。
结果
ATO干预2.5h组,5h组,7.5h组GRP78蛋白的相对表达量较0h组均显著增多(分别是P<0.05,P<0.01,P<0.05)；5h组GRP78蛋白的相对表达量较2.5h组和7.5h组均显著升高(均P<0.05)；而2.5h组GRP78蛋白的相对表达量与7.5h组之间未见明显组间差异(P>0.05)。
ATO干预5h组CHOP蛋白的相对表达量较0h组、2.5h组和7.5h组均显著增多(均P<0.05)；
CHOP siRNA组CHOP mRNA和蛋白的表达量较control siRNA组均显著下降(均P<0.01)；
予以ATO干预后CHOP siRNA组CD4+T细胞的凋亡率为32.39±2.30%,较control siRNA组显著下降(P<0.05)。
结论
(1)三氧化二砷能影响CD4+T细胞中GRP78蛋白和CHOP蛋白的表达；
(2)转染CHOP siRNA能沉默CHOP蛋白的表达,并能部分阻断三氧化二砷诱导的CD4+T细胞的凋亡；
(3)内质网应激-CHOP途径参与三氧化二砷诱导的CD4+T细胞的凋亡。
Chapter1The effect of Arsenic trioxide on airway hyperreactivity and airway inflamation in the acute asthma mouse
Objective
(1Establishing a mouse model of acute asthma, and identify the model;
(2)Observe the effect of ATO (Arsenic Trioxide, ATO) on airway hyperreactivity and airway inflamation in the acute asthma mouse.
Method
Thirty SPF BALB/c female mice were randomly divided into three groups:the normal group(PBS group), the asthma group(OVA/PBS group) and the ATO group(OVA/ATO group), each group contains10mice. Establishing a mouse model of acute asthma after all the mice are raise adaptively for one week according to the following methods. The mice of PBS group were intraperitoneal injected with0.2ml PBS buffer(PH7.2-7.4) at the1st and13th day, and were atomized with6ml PBS buffer(PH7.2-7.4) for30min from the19th to the24th day successively (sum6days). The mice of the PBS group received the intraperitoneal injection of0.2ml PBS buffer(PH7.2-7.4)30min before each atomization. The mice of OVA/PBS group were intraperitoneal injected with0.2ml mixed solution of OVA(ovalbumin, OVA) and aluminium hydroxide gel(10μg OVA and2mg aluminium hydroxide gel were dissolved in PBS buffer thoroughly) at the1st and13th day, and were atomized with6ml5%OVA(Grade V) for30min from the19th to the24th day successively (sum6days). The mice of the OVA/PBS group received the intraperitoneal injection of0.2ml PBS buffer(PH7.2-7.4)30min before each atomization. The mice of OVA/ATO group were intraperitoneal injected with0.2ml mixed solution of OVA and aluminium hydroxide gel(10μg OVA and2mg aluminium hydroxide gel were dissolved in PBS buffer thoroughly) at the1st and13th day, and were atomized with6ml 5%OVA(Grade V) for30min from the19th to the24th day successively (sum6days). The mice of the OVA/ATO group received the intraperitoneal injection of0.2ml ATO solution (2.5mg/kgxmouse weights)30min before each atomization. The mice were sacrificed at25th day, the following characters were detected,(1)the ordinary behavior of the mice,(2)airway hyperreactivity was determined after methacholine challenge by invasive pulmonary impedance method,(3) collecting the BALF and carrying out the cell count and cell classification,(4) making lung pathological slices and observing inflammatory cell infiltration and mucus secretion. All the data was presented with Mean±SD, at first, the data was taken test of normality and homogeneity test of variance. If the data does not conform to normal distribution, the natural logarithmic transformation was conducted. In the case of unequal variances, the Dunnett's T3method was conducted. Analysis of Variance(ANOVA) was conducted for Comparison the difference between multiple sets of mean, and the LSD test was conducted for the pairwise comparison of multiple group data. P<0.05brings the statistics significant.
Result
Prior to the experiment, there is no difference between each group mice in appearance, behavior, the response to stimulation, activity and weight. During the sensitization phase, there is no red, swollen and ulcerate in the injection site of each mice. During the challenge phase, the mice of PBS group feed normally and possess sensitive reflection with clean and smooth fur. The mice of OVA/PBS group displayed exciting, sneezing and scratching in early stage, which displayed crawling and somnolence in the later stage. The mice of OVA/ATO group showed mild symptoms of exciting and sneezing than the OVA/PBS group, and didn't show obvious somnolence. Meanwhile, there was no activity decrease, anorexia and weight decrease occurred in the mice of OVA/ATO group.
Compared with the PBS group, airway responsiveness statistically increased in the OVA/PBS group (P<0.05); airway hyperreactivity (AHR) was eased in the OVA/ATO group compared with that in the OVA/PBS group (P<0.05).
In the BALF of OVA/PBS group, the amount of different cell is list as following, the total of leukocytes is21.11±3.28x104/ml, the Eos is3.10±0.50×104/ml, the Lym is6.60±0.97×104/ml, the Neu is4.92±0.45×104/ml, which are significantly increased (all P<0.01) compared with PBS group. In the OVA/ATO group, the amount of different cell is list as following, the total of leukocytes is13.04±2.58×104/ml, the Eos is1.06±0.19×104/ml, the Lym is2.43±0.28×104/ml, the Neu is2.36±0.29×104/ml, which are significantly decreased (all P<0.01) compared with the OVA/PBS group.
Compared with the PBS group, inflammatory cell infiltration, goblet cell hyperplasia and mucus secretion in lung were far more obvious in OVA/PBS group (P<0.01); airway inflammation and mucus hypersecretion of OVA/ATO group were significantly alleviated than OVA/PBS group (P<0.05).
Conclusion
(1)The mouse model of acute asthma established in this experiment was successful.
(2)Treatment of ATO can alleviate the AHR and airway inflammation of the acute asthma mouse.
Chapter2Arsenic Trioxide promotes the CD4+T cells apoptosis in the acute asthma mouse
Objective
Detecting of the effect of arsenic trioxide on the CD4+T cells apoptosis in the acute asthma mouse.
Method
In vivo:Eighteen SPF BALB/c female mice were randomly divided into three groups:the normal group(PBS group), the asthma group(OVA/PBS group) and the ATO group(OVA/ATO group), each group contains6mice. Establishing a mouse model of acute asthma according the method in the chapter1. The mice were sacrificed at25th day, Splenic CD4+T cells of each group were separated and purified by magnetic cell sorting device (MACS). The number of CD4+T cells of each group was counted. Then CD4+T cells were cultured with RPMI-1640according to the concentration of2×106cell/ml, meanwhile, the ConA(5ug/ml) were added. The CD4+T cells were cultured in the incubator(5%C02,37℃)for24hours. When finishing the culture, flow cytometry(FC) was conducted to detect the apoptosis rate of CD4+T cells.
In vitro:Splenic CD4+T cells of mice in the OVA/PBS group were separated and purified by MACS. Then CD4+T cells were cultured with RPMI-1640according to the concentration of2×106cell/ml, meanwhile, the ConA(5ug/ml) and ATO were added. The CD4+T cells were divided into four groups according to the concentration of the added ATO which including OμM group, luM group,3μM group, and5μM group. All the CD4+T cells were cultured in the incubator(5%CO2,37℃) for20hours. When finishing the culture, flow cytometry(FC) was conducted to detect the apoptosis rate of CD4+T cells. All the data was presented with Mean±SD. At first, the data of each group was taken test of normality and homogeneity test of variance. If the data does not conform to normal distribution, the natural logarithmic transformation was conducted. In the case of unequal variances, the Dunnett's T3method was conducted. Analysis of Variance(ANOVA) was conducted for Comparison the difference between multiple sets of mean, and the LSD test was conducted for the pairwise comparison of multiple group data. P<0.05brings the statistics significant.
Result
In vivo, in the OVA/PBS group, the number of spleen CD4+T cells is (17.88±2.13)×108/L, which is significantly increased when compared with PBS group(P<0.01). The number of the OVA/ATO group is (12.92±2.31)×108/L, which is significantly decreased when compared with OVA/PBS group(P<0.05). In OVA/PBS group, the apoptotic rate of CD4+T cells is20.69±2.68%, which is significantly decreased when compared with PBS group(P<0.05). In OVA/ATO group, the apoptotic rate of CD4+T cells is34.71±0.98%, which is significantly increased when compared with OVA/PBS group(P<0.05).
In vitro, in the3μM group, the apoptotic rate of CD4+T cells is31.85±3.73%, which is significantly increased when compared with OμM group(P<0.05). In the5μM group, the apoptotic rate of CD4+T cells is40.54±1.99%, which is significantly increased when compared with OμM group or3μM group (P<0.01or P<0.05).
Conclusion
Arsenic trioxide promotes the CD4+T cell apoptosis in the acute asthma mouse.
Chapter3Endoplasmic reticulum stress-CHOP pathway involved in the CD4+T cell apoptosis which induced by arsenic trioxide
Objective
(1)Probing the effect of arsenic trioxide on the protein expression of GRP78and CHOP in CD4+T cells.
(2)Expounding the role of CHOP protein in the CD4+T cell apoptosis which induced by arsenic trioxide.
Method
Part one, Splenic CD4+T cells of OVA/PBS group were separated and purified by MACS. The CD4+T cells were cultured with5μM ATO for different time which including Oh,2.5h,5h and7.5h. And the protein expression of GRP78and CHOP were detected.
Part two, Splenic CD4+T cells of OVA/PBS group were divided into CHOP siRNA group and control siRNA group. Transfection of CHOP siRNA can silent the protein expression of CHOP. But the control siRNA doesn't have any silent effection which was used as a negative control. First we transfected the CD4+T cells in CHOP siRNA group with CHOP siRNA and transfected the CD4+T cells in control siRNA group with control siRNA. Then we detected the expression of CHOP mRNA by real-time PCR and the protein expression by western blot to estimate the silent effect of CHOP siRNA. After the successful transfection, the CD4+T cells of both group were cultured with5μM ATO for20hours. Flow cytometry(FC) were conducted to detect the apoptotic rate of CD4+T cells.
All the data was presented with Mean±SD. At first, the data of each group was taken test of normality and homogeneity test of variance. If the data does not conform to normal distribution, the natural logarithmic transformation was conducted. In the case of unequal variances, the Dunnett's T3method was conducted. Independent-samples T test were conducted to compare the difference between two groups. Analysis of Variance(ANOVA) was conducted for comparing the difference between multiple sets of mean, and the LSD test was conducted for the pairwise comparison of multiple group data. P<0.05brings the statistics significant.
Result
The protein expression of GRP78in2.5h group,5h group and7.5h group was significantly increased when compared with Oh group (P<0.05, P<0.01, P<0.05respectively). The protein expression of GRP78in5h group also is more than the2.5h group and7.5h group(both P<0.05). There is no obvious difference between2.5h group and7.5h group(P>0.05).
The protein expression of CHOP in5h group is more than Oh group,2.5h group and7.5h group (all P<0.05).
The mRNA and protein expression of CHOP in CHOP siRNA group both significantly decreased when compared with control siRNA group (both P<0.01).
After cultured with ATO, the CD4+T cell apoptotic rate in CHOP siRNA group is32.39±2.30%, which is significantly decreased when compared with control siRNA group (P<0.05).
Conclusion
(1)Arsenic trioxide effects the protein expression of GRP78and CHOP in CD4+T cells.
(2)Transfection of CHOP siRNA silents the protein expression of CHOP and partially blocks the CD4+T cells apoptosis, which induced by arsenic trioxide.
(3)Endoplasmic reticulum stress-CHOP pathway involved in the CD4+T cell apoptosis which induced by arsenic trioxide.
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