重组腺病毒介导PUMA基因治疗胰腺癌的实验研究
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摘要
胰腺癌是一种高度恶性的肿瘤,其发生发展与多种基因异常有关。胰腺癌手术切除率低,传统的治疗效果差,有待于探讨新的治疗方法。近年来发现,凋亡抑制在胰腺癌的发病机制中起重要作用,因此,通过增加细胞内促凋亡蛋白的表达来诱导细胞凋亡是胰腺癌治疗的有效措施之一。
PUMA是2001年发现的具有较强的促凋亡作用的P53下游基因,是公认的抑癌基因。PUMA在许多恶性肿瘤中表达缺失或低表达,PUMA低表达与恶性肿瘤的发生发展有关,并且恢复细胞内PUMA表达具有明显的肿瘤生长抑制作用。本课题旨在探讨PUMA在胰腺癌中的表达及意义及PUMA基因在胰腺癌中的作用。
第一部分PUMA在胰腺癌组织中的表达及临床意义
目的:研究胰腺癌组织中PUMA蛋白表达与临床病理因素的关系,初步探讨PUMA在胰腺癌发生发展中的作用。
方法:应用免疫组化Envision方法检测60例胰腺导管癌组织石蜡标本中PUMA,Bcl-2和蛋白表达以及19例正常胰腺组织石蜡标本中PUMA蛋白表达,RT-PCR、Western blotting检测进一步证实免疫组化的检测结果。TUNEL检测细胞凋亡,分析PUMA表达与临床病理学指标的关系以及PUMA与AI、P53、Bcl-2表达的相关性。
结果:PUMA在胰腺癌组织中的阳性率为30%(18/60),低于正常胰腺组织90%(17/19),差异有显著性(P=0.002);RT-PCR、Western blotting检测证实了免疫组化结果;PUMA表达与肿瘤大小(P<0.05),淋巴结转移(P<0.05)和远处转移有关(P<0.05),而与肿瘤分化程度和TNM分期无关(分别P>0.05);在PUMA阳性和阴性表达的肿瘤组织中,细胞凋亡指数(AI)分别17.63%±6.27%和13.44%±5.86%,差异有显著性(p<0.05);在PUMA阴性表达的肿瘤组织中的细胞凋亡指数明显低于正常胰腺组织(16.12 %±5.72 %),差异有显著性(p<0.05);P53和Bcl-2在胰腺癌中的表达率分别为46.7%(28/60)和41.7%(25/60),PUMA与P53和Bcl-2表达分别有显著的负相关性(p=0.013和P=0.046)。
结论:胰腺癌细胞凋亡抑制及肿瘤转移与PUMA蛋白表达缺失有关,PUMA可能是胰腺癌预后的判断指标和基因治疗的靶点。
第二部分胰腺癌细胞CAR受体表达与5型腺病毒转导效率的关系
目的:通过体外、体内实验探讨胰腺癌肿瘤细胞CAR表达水平与腺病毒转导效率的关系,为腺病毒相关的生物制剂在胰腺癌患者个体化应用提供实验依据。
方法:先将载有绿色荧光蛋白的Ad5型腺病毒(Ad-GFP)以100MOI分别感染人胰腺癌细胞系AsPC-1、CaPan-1、BxPC-3、Panc-1细胞6～96h,再以20～1000 MOI的Ad-GFP感染AsPC-1、CaPan-1、BxPC-3、Panc-1细胞24 h。感染结束后分别通过流式细胞术检测Ad-GFP在不同细胞系的转导效率。采用Western blotting、免疫组化方法检测这些细胞中CAR的表达水平。将CaPan-1、BxPC-3细胞分别接种于裸鼠背部,接种细胞数分别为5×106,建立裸鼠移植瘤模型。待肿瘤长径达10mm时,在裸鼠移植瘤内注射Ad-GFP 1×108 pfu.试验分2组,第一组:1-14天内动态观察肿瘤内荧光的变化,第二组:3天处死裸鼠,剖取瘤组织,荧光显微镜观察冰冻切片中GFP的表达情况以判定腺病毒在瘤体内的转导效率,同时western blot法检测瘤组织内的CAR表达水平。
结果:4种细胞的转染效率在24小时最高,AsPC-1、CaPan-1细胞在MOI50时,转染效率近100%,Panc-1细胞在200MOI时转染效率接近100%,而BxPC-3在MOI1000时,转染效率为60%。各种细胞的CAR蛋白表达水平与腺病毒的转导效率呈正相关。在注射Ad-GFP的裸鼠移植瘤组织中,3天可见CaPan-1瘤组织内有明显的绿色荧光,而BxPC-3瘤组织内荧光强度少于前者;14天后荧光完全消失。冰冻切片发现CaPan-1细胞内明显点状荧光,而BxPC-3细胞荧光较弱。CAR在CaPan-1移植瘤组织的表达高于BxPC-3移植瘤组织,表明瘤体内CAR表达水平与Ad-GFP的转导效率也呈正相关。
结论:体内外实验均显示肿瘤细胞的CAR表达水平与5型腺病毒转导效率密切相关,胰腺癌患者治疗前检测组织中CAR表达水平有助于规范腺病毒载体的基因治疗药物个体化使用
第三部分携带PUMA基因的重组腺病毒(Ad-PUMA)的构建及制备
目的:构建含有人PUMA的重组腺病毒,为下一步转染人胰腺癌细胞研究奠定基础。
方法:以重组质粒pCEP4-PUMA中提取的PUMA基因为模板,采用PCR法扩增基因片段。将扩增的基因片段插入穿梭质粒pShuttle-CMV,并转化大肠肝菌BJ-5183。筛选重组质粒pShuttle-CMV-PUMA,与pAdEasy-1一起电转化BJ5183细胞。筛选重组腺病毒质粒pAdEasy-PUMA,用Lipofectamine转染293细胞,制备携带人PUMA基因的重组复制缺陷型腺病毒(Ad-PUMA)。氯化铯密度梯度离心法纯化Ad-PUMA病毒颗粒,并测定病毒滴度。
结果①经限制性内切酶酶切,证实Ad-PUMA构建成功②限制性内切酶酶切确定穿梭质粒重组于病毒骨架;显微镜下观察HEK293细胞形态,证实病毒包装复制成功③病毒滴度2.032×1010pfu/ml,达到进一步体内、外实验要求。
结论:成功构建了重组腺病毒Ad-PUMA,为了解PUMA在胰腺癌中的治疗作用奠定基础。
第四部分Ad-PUMA治疗胰腺癌的体外研究
目的:探讨Ad-PUMA对体外胰腺癌细胞的凋亡促进作用和生长抑制作用.
方法:以不同MOI值的Ad-PUMA作用AsPC-1、CaPan-1、BxPC-3、Panc-1细胞,MTT检测PUMA对细胞增殖的影响,PI染色、DNA Ladder、流式细胞仪检测PUMA对细胞凋亡的影响;Western blotting检测细胞内PUMA蛋白表达的变化以及Bax、Bcl-2、细胞色素C、Caspase-9,3表达的影响,流式细胞仪检测细胞内Caspase活性
结果: Ad-PUMA抑制胰腺癌细胞系细胞增殖和生长,并且PUMA抑制胰腺癌细胞生长表现出时间依赖性和剂量依赖性,但PUMA对不同胰腺癌细胞的生长抑制作用并不相同,在相同条件下,Aspc-1细胞的生长抑制最明显,而BxPC-3的生长抑制不明显。DNA Ladder、FCM、PI染色分析检测到明显的Ad-PUMA诱导的细胞凋亡。Western blotting发现,在PUMA表达上调的同时,细胞内Bax、Bcl-2、细胞色素C、Caspases蛋白表达也随着变化,caspases酶的活性明显提高。
结论:PUMA通过促进凋亡而抑制胰腺癌细胞生长,PUMA通过线粒体途径发挥作用.
第五部分Ad-PUMA治疗胰腺癌的体内研究
目的:探讨Ad-PUMA对体内胰腺癌细胞的生长抑制作用。
方法:建立裸鼠AsPC-1胰腺癌模型,待瘤体生长到10mm左右。治疗前一天,移植肿瘤局部注射Ad-PUMA1×108PUF/ml/50ul,第3d注射第二次,第6 d注射第3次,从治疗前一天开始测量肿瘤体积,每2d测量一次至第21天。在治疗21天,TUNEL检测细胞凋亡、免疫组化检测ki67了解细胞增殖,western blotting和RT-PCR检测细胞PUMA表达的变化。
结果:在实验组,肿瘤生长明显减慢,21天后的肿瘤的体积明显低于对照组,而细胞内PUMA表达明显高于对照组;实验组细胞增殖受抑制,细胞凋亡指数增加。
结论: Ad-PUMA转染促进体内肿瘤细胞凋亡并抑制其生长。
第六部分Ad-PUMA联合吉西他滨治疗胰腺癌的体内研究
目的:探讨Ad-PUMA联合吉西他滨对体内外胰腺癌细胞生长的影响。
方法:将AsPC-1细胞分别暴露于系列浓度的吉西他滨或系列浓度吉他西滨联合5MOI Ad-PUMA 48h,MTT法测定细胞的存活率。按照上述方法建立裸鼠AsPC-1胰腺癌模型,待瘤体生长到10mm左右,吉西他滨、Ad-PUMA或两者联合治疗移植瘤。于0、3、6天,肿瘤局部注射Ad-PUMA 1×109PUF/ml/50ul,吉他西滨的给药方式为腹腔注射,剂量为100mg/kg,于0、3、6天给药。从治疗前一天开始测量肿瘤体积,2d测量一次至21天。
结果:吉他西滨以剂量依赖方式抑制AsPC-1细胞增殖,但吉他西滨联合Ad-PUMA后对AsPC-1细胞增殖的抑制更加明显,联合后的IC50为(0.0076±0.012)μg/ml,其敏感性较单独吉西他滨作用增加6.9倍。单独以Ad-PUMA治疗,肿瘤生长抑制率为62.7%,单独吉西他滨治疗时,肿瘤生长抑制率为43.2%,联合后的抑制率为82% (P<0.01)。
结论:PUMA基因转染增强胰腺癌细胞对吉西他滨的化疗敏感性,增强抗肿瘤效果。
The incidence of pancreatic cancer is increasing,so far neither an early diagnosis nor a therapeutic strategy for advanced lesions has been developed.It is an urgent mission for both the clinicians and the scientists who are challenging pancreatic cancer to find a breakthrough using new technologies.Apoptosis, also called programmed cell death, is a genetically encoded program that disposes of unwanted cells. Disruptions of this pathway have been implicated as a cause of cancer. Therefore, genetic restoration of the apoptotic pathway or introduction of pro-apoptotic molecules is an attractive approach for treating tumors, including malignant pancreatic cancer. Recently, p53 upregulated modulator of apoptosis (PUMA) was identified as a p53-inducible pro-apoptotic molecule,. PUMA shares homology with Bcl-2 family proteins within a short stretch of amino acids termed the BH3 domain, a region that allows interaction among the family members. Proteins with similar homology, called BH3-only proteins, constitute the third subgroup of the Bcl-2 family and include Bad, Bid, Bik, and Bim .These proteins play an important role in apoptosis induction.It has been reported PUMA is lost or lower expression in many cancer cells,which is significantly related to tumorgenesis.The therapeutic possibility of PUMA as a gene target in pancreatic cancer was evaluated.
Pant one The expression and biological function of PUMA in pancreatic carcinoma
OBJECTIVE: To investigate the expression of PUMA in pancreatic cancers(PC)tissue and its correlations to clinicopathologic features and carcinogenesis
METHODS:The expression of PUMA ,P53and bcl-2 in 60 specimens of PC were assessed by immunohistochemical staining Envision method ,so was PUMA in 19 speciments of no-cancer pancreatic tissues,PUMA gene was assessed by RT-PCR and Western blot method in 5 cases of newly resected PC tissues and normal PC.Apoptosis index(AI) was determinded with TUNEL method; PUMA correlations to p53、bcl-2、AI and clinicopathologic factors were analyzed .
RESULTS: The positive rates of PUMA was significantly lower in PC 30%(18/60)than in no-cancer pancreatic tissues90%(17/19()p<0.01), The results of RT-PCR and Western blot verified the results of PUMA expression by IP analysis.PUMA expression in the tumor was correlated with the tumor size (p<0.05) and lymph node (P<0.05) and distant metastasis (P<0.05). No relationship to any other clinical or pathological features were found. The AI was 17.63%±6.27%, 13.44%±5.86% in cancer tissues of positive and negative expression of PUMA, respectively (P < 0.05). the AI in cancer tissues of negative expression of PUMA was lower than that of in normal pancreatic tissues 16.12 %±5.72 %,( p<0.05 ) .The positive rate of p53 and bcl-2 in PC were 46.7%(28/60) and 41.7%(25/60).Spearman rank correlation coefficient test revealed a significant inverse association for PUMA and bcl-2 , PUMA and p53 expression (P<0.05, respectively)
CONCLUSION: The expression of PUMA is lost or lower in pancreatic cancer, which may be involved in the oncogenesis development and lymph node and distant metastasis,PUMA may be as a new target for the therapy of pancreatic cancer.
Pant two Relationship between coxsackie adenovirus receptor expression levels and the ad5 transduction eficiency in pancreatic cancer
0bjective : To investigate the relationship between coxsackie adenovirus receptor(CAR)expression and the eficiency of adenovirus gene transfer,in an effort to provide evidence for the clinical application of adenovirus-related Biopreparations.
Methods:The pancreatic cancer cell lines(AsPC-1、CaPan-1、BxPC-3、Panc-1) were infected with Ad-GFP(100 MOI) labeled by immunofluorescence:0-96h later we measured the Ad-GFP transduction eficiencies in the above cell lines by flow cytometric analysis,and Ad-GFP(0-1000MOI) infected cells obove for 24h, transduction eficiencies was measured.CAR expression levels in vitro were assayed by Western-blotting and IP.CaPan-1、BxPC-3(5×106)cells were implanted subcutaneously into nude mice.When the tumor sizes reached 10 mm in diameter,the xenografts were injected with Ad-GFP(1×108pfu).During 1-14 days, GFP wsa observed by DCC-3 camara in the first groups,and in another group ,after 72h the mice were killed and the GFP expression were observed by fluorescence microscopy to determine the transduction efficiency.CAR expression was detected by Western blotting in the xenograft tissues.
Results:We found that Ad transduction efficiencies varied greatly in the pancreatic ce11s of the same tissue 0rigin.The transduction efficiencies in AsPC-1、CaPan-1 (100%,50 MOI), Panc-1(100%,MOI200), BxPC-3(60%,1000MOI) was different.Consistently,GFP expression was much higher in exografts sections resulting from CaPan-1 than thatfrom BxPC-3.Increased CAR expression was predictive for more efficient gene transfer in vitro an d in vivo.
Conclusion:CAR expression level is closely correlated to Ad transduction efficiency,which suggests that measurement of CAR expression in tumor tissues is useful for individualized adenovirus based gene therapy in clinic.
Part three Construction and Identification of Recombinant Adenovirus for Expression of PUMA gene
Objective To construct a recombinant adenovirus for expression of PUMA gene.
Methods Amplify gene fragment by PCR using the PUMA gene extracted from recombinant plasmid pHA-PUMA as a template insert into shuttle plasmid pShuttle-CMV and transform to E coli DH 5a.Screen recombinant plasmid pShuttle-CMV-PUMA and transform to competent BJ5183 cells previously transformed witll pAdEasy-1.Screen recombinant plasmid pAdEasy-PUMA and transfect to 293 cels in mediation of Lipofectamine to prepare replication-deficient adenovirus Ad-PUMA carrying PUMA gene.Purify the prepared
Ad-PUMA virus particles by density gradient centrifugation with cesium chloride and determine the titer.
Results Recombinants were selected by kanamycin resistence and confirmed by PacI.The restrictive endonuclease analysis confirmed that correct recombinant adenovirus plasmid Was constructed(30 kb and 4.5 kb fragments were dotained after PacI digestion). Twenty four hours after transfection,the fluorescence Was observed in 293 cels,and the titer of purified Ad-PUMA was 2.032×1010pfu/ml
Conclusion Recombinant adenovirus Ad-PUMA for expression of PUMA was successfully constructed ,which provided a basis for gene therapy of tumors.
Part four Expremental study of Ad-PUMA on pancreatic cancer in vitro
Objective To study the effect of recombinant adnenovirus vector mediated PUMA(Ad-PUMA)on pancreatic cancer cells (PC)
Methods AsPC-1、CaPan-1、BxPC-3、Panc-1 cells were infected with Ad-PUMA, respectively. The inhibition rate of PC were examined by MTT,the apoptosis was checked by FCM,PI staining and DNA Ladder. Western blotting was used to examine the expression of PUMA and its downstreaming Bax、Bcl-2、cytochrome c、Caspases-9,3, FCM was used to examine the activity of caspases. Results: The Proliferation rate of PC cell lines were suppressed significantly by Ad-PUMA infection in a viral dose-dependent and time-dependent manner,and significant cell apoptosis was checked through DNA Ladder,FCM,PI staining. The expression of PUMA protein increased with titer of Ad-PUMA, they were all does-dependant and time-dependant.With the PUMA upregulation, Bax、Bcl-2、cytochrome c、Caspases-9,3 protein in cells changed too.The activity of caspase-9,3 increased significantly.
Conclusions Recombimant adenoviral-mediated PUMA gene increase the cell-killing effect by improve the cell apoptosis,which is through the way of mitochondrion.
Part five Experimental study of Ad-PUMA on pancreatic cancer in vito
Objective To study the effect of Ad-PUMA in vito
Methods To establish tumors for model system,5×106AsPC-1 cells were injected subcutaneously onto the flanks of the femals SCID-Bg mice.After approximately 21-28ds,palpable tumors developed.These animals were then treated with Ad-PUMA,Ad-GFP and injection of PBS.At 0,3,6 days,BPS(50ul), Ad-GFP and Ad-PUMA plasmids(3×108PUF/ml/50ul)was injected to the tumor. Tumor growth at different time points was record in 21 days,and after 21d,and the apoptosis index and ki67 were examined by TUNEL and IP,the expression of PUMA and PUMA mRNA protein were examine by western blotting and RT-PCR method.
Results Ad-PUMA had a dramatic effect on tumor volume in the first 14days following injection,this effect diminished over time ,but still resulted in a 44.2 %(P<0.05)decrease in tumor after 21 days.Treatment with the Ad-GFP control virus did not substantially alter tumor in this model system(p>0.05).The apoptosis indix in Ad-PUMA groups was higher than than of in control groups,and the ki-67 expression was lower in Ad-PUMA groups.The expression of PUMA and PUMA-mRNA was much higher in Ad-PUMA groups than other groups (p<0.05).
Conclusions Recombimant adenoviral-mediated PUMA inhibited growth of tumor in vitro.
Part six Experimental study of gemcitabine combined with Ad-PUMA on pancreatic cancer in vito
Objective To study the effect of recombinant adnenovirus vector mediated PUMA combined with gemcitabine on pancreatic cancer in vitro and vivo .
Methods:Pancreatic cancer cells AsPC-1 transfected by PUMA(5MOI) or not reseparately treated with serial concentrations of gemcitabine. The sensitivity of cells to gemcitabine was determined by MTT assay. To establish tumors for model system as part five. These animals were then treated with Ad-PUMA (1×109puf/ml/50ul)at0,3,6days,along with viral injection,animals recived a 100ul intraperitoneal injection of gemcitabine(100mg/kg) on days0,3,6,Ad-GFP was as the control. Tumor growth at different points was record in 21d
Results Gemcitabine inhibited AsPC-1 cell proliferation in a dose dependent manner,but the cell proliferation was more significant in gemcitabine combined with PUMA expression clones than in parental AsPC-1cells.The IC50(0.0076±0.012)μg/ml in combined groups increased 6.9 times than that of IC50(0.053±0.001)μg/ml in gemcitabine groups. When Ad-PUMA or gemecitabine was injected as a single agent it resultd in reduction in 62.75(P<0.01 vs control) or 42.3% (p<0.05 vs control)tumor growth ,however, the combination of Ad-PUMA with gemcitabine reduced tumor volume by more than 82%(P<0.001 vs control)on day 21.
Conclusion PUMA significantly enhances chemosensitivity of AsPC-1 to gemcitabine, and PUMA and gemcitabine exert anadditive anti-tumor effect in vivo.
PUMA是2001年发现的具有较强的促凋亡作用的P53下游基因,是公认的抑癌基因。PUMA在许多恶性肿瘤中表达缺失或低表达,PUMA低表达与恶性肿瘤的发生发展有关,并且恢复细胞内PUMA表达具有明显的肿瘤生长抑制作用。本课题旨在探讨PUMA在胰腺癌中的表达及意义及PUMA基因在胰腺癌中的作用。
第一部分PUMA在胰腺癌组织中的表达及临床意义
目的:研究胰腺癌组织中PUMA蛋白表达与临床病理因素的关系,初步探讨PUMA在胰腺癌发生发展中的作用。
方法:应用免疫组化Envision方法检测60例胰腺导管癌组织石蜡标本中PUMA,Bcl-2和蛋白表达以及19例正常胰腺组织石蜡标本中PUMA蛋白表达,RT-PCR、Western blotting检测进一步证实免疫组化的检测结果。TUNEL检测细胞凋亡,分析PUMA表达与临床病理学指标的关系以及PUMA与AI、P53、Bcl-2表达的相关性。
结果:PUMA在胰腺癌组织中的阳性率为30%(18/60),低于正常胰腺组织90%(17/19),差异有显著性(P=0.002);RT-PCR、Western blotting检测证实了免疫组化结果;PUMA表达与肿瘤大小(P<0.05),淋巴结转移(P<0.05)和远处转移有关(P<0.05),而与肿瘤分化程度和TNM分期无关(分别P>0.05);在PUMA阳性和阴性表达的肿瘤组织中,细胞凋亡指数(AI)分别17.63%±6.27%和13.44%±5.86%,差异有显著性(p<0.05);在PUMA阴性表达的肿瘤组织中的细胞凋亡指数明显低于正常胰腺组织(16.12 %±5.72 %),差异有显著性(p<0.05);P53和Bcl-2在胰腺癌中的表达率分别为46.7%(28/60)和41.7%(25/60),PUMA与P53和Bcl-2表达分别有显著的负相关性(p=0.013和P=0.046)。
结论:胰腺癌细胞凋亡抑制及肿瘤转移与PUMA蛋白表达缺失有关,PUMA可能是胰腺癌预后的判断指标和基因治疗的靶点。
第二部分胰腺癌细胞CAR受体表达与5型腺病毒转导效率的关系
目的:通过体外、体内实验探讨胰腺癌肿瘤细胞CAR表达水平与腺病毒转导效率的关系,为腺病毒相关的生物制剂在胰腺癌患者个体化应用提供实验依据。
方法:先将载有绿色荧光蛋白的Ad5型腺病毒(Ad-GFP)以100MOI分别感染人胰腺癌细胞系AsPC-1、CaPan-1、BxPC-3、Panc-1细胞6～96h,再以20～1000 MOI的Ad-GFP感染AsPC-1、CaPan-1、BxPC-3、Panc-1细胞24 h。感染结束后分别通过流式细胞术检测Ad-GFP在不同细胞系的转导效率。采用Western blotting、免疫组化方法检测这些细胞中CAR的表达水平。将CaPan-1、BxPC-3细胞分别接种于裸鼠背部,接种细胞数分别为5×106,建立裸鼠移植瘤模型。待肿瘤长径达10mm时,在裸鼠移植瘤内注射Ad-GFP 1×108 pfu.试验分2组,第一组:1-14天内动态观察肿瘤内荧光的变化,第二组:3天处死裸鼠,剖取瘤组织,荧光显微镜观察冰冻切片中GFP的表达情况以判定腺病毒在瘤体内的转导效率,同时western blot法检测瘤组织内的CAR表达水平。
结果:4种细胞的转染效率在24小时最高,AsPC-1、CaPan-1细胞在MOI50时,转染效率近100%,Panc-1细胞在200MOI时转染效率接近100%,而BxPC-3在MOI1000时,转染效率为60%。各种细胞的CAR蛋白表达水平与腺病毒的转导效率呈正相关。在注射Ad-GFP的裸鼠移植瘤组织中,3天可见CaPan-1瘤组织内有明显的绿色荧光,而BxPC-3瘤组织内荧光强度少于前者;14天后荧光完全消失。冰冻切片发现CaPan-1细胞内明显点状荧光,而BxPC-3细胞荧光较弱。CAR在CaPan-1移植瘤组织的表达高于BxPC-3移植瘤组织,表明瘤体内CAR表达水平与Ad-GFP的转导效率也呈正相关。
结论:体内外实验均显示肿瘤细胞的CAR表达水平与5型腺病毒转导效率密切相关,胰腺癌患者治疗前检测组织中CAR表达水平有助于规范腺病毒载体的基因治疗药物个体化使用
第三部分携带PUMA基因的重组腺病毒(Ad-PUMA)的构建及制备
目的:构建含有人PUMA的重组腺病毒,为下一步转染人胰腺癌细胞研究奠定基础。
方法:以重组质粒pCEP4-PUMA中提取的PUMA基因为模板,采用PCR法扩增基因片段。将扩增的基因片段插入穿梭质粒pShuttle-CMV,并转化大肠肝菌BJ-5183。筛选重组质粒pShuttle-CMV-PUMA,与pAdEasy-1一起电转化BJ5183细胞。筛选重组腺病毒质粒pAdEasy-PUMA,用Lipofectamine转染293细胞,制备携带人PUMA基因的重组复制缺陷型腺病毒(Ad-PUMA)。氯化铯密度梯度离心法纯化Ad-PUMA病毒颗粒,并测定病毒滴度。
结果①经限制性内切酶酶切,证实Ad-PUMA构建成功②限制性内切酶酶切确定穿梭质粒重组于病毒骨架;显微镜下观察HEK293细胞形态,证实病毒包装复制成功③病毒滴度2.032×1010pfu/ml,达到进一步体内、外实验要求。
结论:成功构建了重组腺病毒Ad-PUMA,为了解PUMA在胰腺癌中的治疗作用奠定基础。
第四部分Ad-PUMA治疗胰腺癌的体外研究
目的:探讨Ad-PUMA对体外胰腺癌细胞的凋亡促进作用和生长抑制作用.
方法:以不同MOI值的Ad-PUMA作用AsPC-1、CaPan-1、BxPC-3、Panc-1细胞,MTT检测PUMA对细胞增殖的影响,PI染色、DNA Ladder、流式细胞仪检测PUMA对细胞凋亡的影响;Western blotting检测细胞内PUMA蛋白表达的变化以及Bax、Bcl-2、细胞色素C、Caspase-9,3表达的影响,流式细胞仪检测细胞内Caspase活性
结果: Ad-PUMA抑制胰腺癌细胞系细胞增殖和生长,并且PUMA抑制胰腺癌细胞生长表现出时间依赖性和剂量依赖性,但PUMA对不同胰腺癌细胞的生长抑制作用并不相同,在相同条件下,Aspc-1细胞的生长抑制最明显,而BxPC-3的生长抑制不明显。DNA Ladder、FCM、PI染色分析检测到明显的Ad-PUMA诱导的细胞凋亡。Western blotting发现,在PUMA表达上调的同时,细胞内Bax、Bcl-2、细胞色素C、Caspases蛋白表达也随着变化,caspases酶的活性明显提高。
结论:PUMA通过促进凋亡而抑制胰腺癌细胞生长,PUMA通过线粒体途径发挥作用.
第五部分Ad-PUMA治疗胰腺癌的体内研究
目的:探讨Ad-PUMA对体内胰腺癌细胞的生长抑制作用。
方法:建立裸鼠AsPC-1胰腺癌模型,待瘤体生长到10mm左右。治疗前一天,移植肿瘤局部注射Ad-PUMA1×108PUF/ml/50ul,第3d注射第二次,第6 d注射第3次,从治疗前一天开始测量肿瘤体积,每2d测量一次至第21天。在治疗21天,TUNEL检测细胞凋亡、免疫组化检测ki67了解细胞增殖,western blotting和RT-PCR检测细胞PUMA表达的变化。
结果:在实验组,肿瘤生长明显减慢,21天后的肿瘤的体积明显低于对照组,而细胞内PUMA表达明显高于对照组;实验组细胞增殖受抑制,细胞凋亡指数增加。
结论: Ad-PUMA转染促进体内肿瘤细胞凋亡并抑制其生长。
第六部分Ad-PUMA联合吉西他滨治疗胰腺癌的体内研究
目的:探讨Ad-PUMA联合吉西他滨对体内外胰腺癌细胞生长的影响。
方法:将AsPC-1细胞分别暴露于系列浓度的吉西他滨或系列浓度吉他西滨联合5MOI Ad-PUMA 48h,MTT法测定细胞的存活率。按照上述方法建立裸鼠AsPC-1胰腺癌模型,待瘤体生长到10mm左右,吉西他滨、Ad-PUMA或两者联合治疗移植瘤。于0、3、6天,肿瘤局部注射Ad-PUMA 1×109PUF/ml/50ul,吉他西滨的给药方式为腹腔注射,剂量为100mg/kg,于0、3、6天给药。从治疗前一天开始测量肿瘤体积,2d测量一次至21天。
结果:吉他西滨以剂量依赖方式抑制AsPC-1细胞增殖,但吉他西滨联合Ad-PUMA后对AsPC-1细胞增殖的抑制更加明显,联合后的IC50为(0.0076±0.012)μg/ml,其敏感性较单独吉西他滨作用增加6.9倍。单独以Ad-PUMA治疗,肿瘤生长抑制率为62.7%,单独吉西他滨治疗时,肿瘤生长抑制率为43.2%,联合后的抑制率为82% (P<0.01)。
结论:PUMA基因转染增强胰腺癌细胞对吉西他滨的化疗敏感性,增强抗肿瘤效果。
The incidence of pancreatic cancer is increasing,so far neither an early diagnosis nor a therapeutic strategy for advanced lesions has been developed.It is an urgent mission for both the clinicians and the scientists who are challenging pancreatic cancer to find a breakthrough using new technologies.Apoptosis, also called programmed cell death, is a genetically encoded program that disposes of unwanted cells. Disruptions of this pathway have been implicated as a cause of cancer. Therefore, genetic restoration of the apoptotic pathway or introduction of pro-apoptotic molecules is an attractive approach for treating tumors, including malignant pancreatic cancer. Recently, p53 upregulated modulator of apoptosis (PUMA) was identified as a p53-inducible pro-apoptotic molecule,. PUMA shares homology with Bcl-2 family proteins within a short stretch of amino acids termed the BH3 domain, a region that allows interaction among the family members. Proteins with similar homology, called BH3-only proteins, constitute the third subgroup of the Bcl-2 family and include Bad, Bid, Bik, and Bim .These proteins play an important role in apoptosis induction.It has been reported PUMA is lost or lower expression in many cancer cells,which is significantly related to tumorgenesis.The therapeutic possibility of PUMA as a gene target in pancreatic cancer was evaluated.
Pant one The expression and biological function of PUMA in pancreatic carcinoma
OBJECTIVE: To investigate the expression of PUMA in pancreatic cancers(PC)tissue and its correlations to clinicopathologic features and carcinogenesis
METHODS:The expression of PUMA ,P53and bcl-2 in 60 specimens of PC were assessed by immunohistochemical staining Envision method ,so was PUMA in 19 speciments of no-cancer pancreatic tissues,PUMA gene was assessed by RT-PCR and Western blot method in 5 cases of newly resected PC tissues and normal PC.Apoptosis index(AI) was determinded with TUNEL method; PUMA correlations to p53、bcl-2、AI and clinicopathologic factors were analyzed .
RESULTS: The positive rates of PUMA was significantly lower in PC 30%(18/60)than in no-cancer pancreatic tissues90%(17/19()p<0.01), The results of RT-PCR and Western blot verified the results of PUMA expression by IP analysis.PUMA expression in the tumor was correlated with the tumor size (p<0.05) and lymph node (P<0.05) and distant metastasis (P<0.05). No relationship to any other clinical or pathological features were found. The AI was 17.63%±6.27%, 13.44%±5.86% in cancer tissues of positive and negative expression of PUMA, respectively (P < 0.05). the AI in cancer tissues of negative expression of PUMA was lower than that of in normal pancreatic tissues 16.12 %±5.72 %,( p<0.05 ) .The positive rate of p53 and bcl-2 in PC were 46.7%(28/60) and 41.7%(25/60).Spearman rank correlation coefficient test revealed a significant inverse association for PUMA and bcl-2 , PUMA and p53 expression (P<0.05, respectively)
CONCLUSION: The expression of PUMA is lost or lower in pancreatic cancer, which may be involved in the oncogenesis development and lymph node and distant metastasis,PUMA may be as a new target for the therapy of pancreatic cancer.
Pant two Relationship between coxsackie adenovirus receptor expression levels and the ad5 transduction eficiency in pancreatic cancer
0bjective : To investigate the relationship between coxsackie adenovirus receptor(CAR)expression and the eficiency of adenovirus gene transfer,in an effort to provide evidence for the clinical application of adenovirus-related Biopreparations.
Methods:The pancreatic cancer cell lines(AsPC-1、CaPan-1、BxPC-3、Panc-1) were infected with Ad-GFP(100 MOI) labeled by immunofluorescence:0-96h later we measured the Ad-GFP transduction eficiencies in the above cell lines by flow cytometric analysis,and Ad-GFP(0-1000MOI) infected cells obove for 24h, transduction eficiencies was measured.CAR expression levels in vitro were assayed by Western-blotting and IP.CaPan-1、BxPC-3(5×106)cells were implanted subcutaneously into nude mice.When the tumor sizes reached 10 mm in diameter,the xenografts were injected with Ad-GFP(1×108pfu).During 1-14 days, GFP wsa observed by DCC-3 camara in the first groups,and in another group ,after 72h the mice were killed and the GFP expression were observed by fluorescence microscopy to determine the transduction efficiency.CAR expression was detected by Western blotting in the xenograft tissues.
Results:We found that Ad transduction efficiencies varied greatly in the pancreatic ce11s of the same tissue 0rigin.The transduction efficiencies in AsPC-1、CaPan-1 (100%,50 MOI), Panc-1(100%,MOI200), BxPC-3(60%,1000MOI) was different.Consistently,GFP expression was much higher in exografts sections resulting from CaPan-1 than thatfrom BxPC-3.Increased CAR expression was predictive for more efficient gene transfer in vitro an d in vivo.
Conclusion:CAR expression level is closely correlated to Ad transduction efficiency,which suggests that measurement of CAR expression in tumor tissues is useful for individualized adenovirus based gene therapy in clinic.
Part three Construction and Identification of Recombinant Adenovirus for Expression of PUMA gene
Objective To construct a recombinant adenovirus for expression of PUMA gene.
Methods Amplify gene fragment by PCR using the PUMA gene extracted from recombinant plasmid pHA-PUMA as a template insert into shuttle plasmid pShuttle-CMV and transform to E coli DH 5a.Screen recombinant plasmid pShuttle-CMV-PUMA and transform to competent BJ5183 cells previously transformed witll pAdEasy-1.Screen recombinant plasmid pAdEasy-PUMA and transfect to 293 cels in mediation of Lipofectamine to prepare replication-deficient adenovirus Ad-PUMA carrying PUMA gene.Purify the prepared
Ad-PUMA virus particles by density gradient centrifugation with cesium chloride and determine the titer.
Results Recombinants were selected by kanamycin resistence and confirmed by PacI.The restrictive endonuclease analysis confirmed that correct recombinant adenovirus plasmid Was constructed(30 kb and 4.5 kb fragments were dotained after PacI digestion). Twenty four hours after transfection,the fluorescence Was observed in 293 cels,and the titer of purified Ad-PUMA was 2.032×1010pfu/ml
Conclusion Recombinant adenovirus Ad-PUMA for expression of PUMA was successfully constructed ,which provided a basis for gene therapy of tumors.
Part four Expremental study of Ad-PUMA on pancreatic cancer in vitro
Objective To study the effect of recombinant adnenovirus vector mediated PUMA(Ad-PUMA)on pancreatic cancer cells (PC)
Methods AsPC-1、CaPan-1、BxPC-3、Panc-1 cells were infected with Ad-PUMA, respectively. The inhibition rate of PC were examined by MTT,the apoptosis was checked by FCM,PI staining and DNA Ladder. Western blotting was used to examine the expression of PUMA and its downstreaming Bax、Bcl-2、cytochrome c、Caspases-9,3, FCM was used to examine the activity of caspases. Results: The Proliferation rate of PC cell lines were suppressed significantly by Ad-PUMA infection in a viral dose-dependent and time-dependent manner,and significant cell apoptosis was checked through DNA Ladder,FCM,PI staining. The expression of PUMA protein increased with titer of Ad-PUMA, they were all does-dependant and time-dependant.With the PUMA upregulation, Bax、Bcl-2、cytochrome c、Caspases-9,3 protein in cells changed too.The activity of caspase-9,3 increased significantly.
Conclusions Recombimant adenoviral-mediated PUMA gene increase the cell-killing effect by improve the cell apoptosis,which is through the way of mitochondrion.
Part five Experimental study of Ad-PUMA on pancreatic cancer in vito
Objective To study the effect of Ad-PUMA in vito
Methods To establish tumors for model system,5×106AsPC-1 cells were injected subcutaneously onto the flanks of the femals SCID-Bg mice.After approximately 21-28ds,palpable tumors developed.These animals were then treated with Ad-PUMA,Ad-GFP and injection of PBS.At 0,3,6 days,BPS(50ul), Ad-GFP and Ad-PUMA plasmids(3×108PUF/ml/50ul)was injected to the tumor. Tumor growth at different time points was record in 21 days,and after 21d,and the apoptosis index and ki67 were examined by TUNEL and IP,the expression of PUMA and PUMA mRNA protein were examine by western blotting and RT-PCR method.
Results Ad-PUMA had a dramatic effect on tumor volume in the first 14days following injection,this effect diminished over time ,but still resulted in a 44.2 %(P<0.05)decrease in tumor after 21 days.Treatment with the Ad-GFP control virus did not substantially alter tumor in this model system(p>0.05).The apoptosis indix in Ad-PUMA groups was higher than than of in control groups,and the ki-67 expression was lower in Ad-PUMA groups.The expression of PUMA and PUMA-mRNA was much higher in Ad-PUMA groups than other groups (p<0.05).
Conclusions Recombimant adenoviral-mediated PUMA inhibited growth of tumor in vitro.
Part six Experimental study of gemcitabine combined with Ad-PUMA on pancreatic cancer in vito
Objective To study the effect of recombinant adnenovirus vector mediated PUMA combined with gemcitabine on pancreatic cancer in vitro and vivo .
Methods:Pancreatic cancer cells AsPC-1 transfected by PUMA(5MOI) or not reseparately treated with serial concentrations of gemcitabine. The sensitivity of cells to gemcitabine was determined by MTT assay. To establish tumors for model system as part five. These animals were then treated with Ad-PUMA (1×109puf/ml/50ul)at0,3,6days,along with viral injection,animals recived a 100ul intraperitoneal injection of gemcitabine(100mg/kg) on days0,3,6,Ad-GFP was as the control. Tumor growth at different points was record in 21d
Results Gemcitabine inhibited AsPC-1 cell proliferation in a dose dependent manner,but the cell proliferation was more significant in gemcitabine combined with PUMA expression clones than in parental AsPC-1cells.The IC50(0.0076±0.012)μg/ml in combined groups increased 6.9 times than that of IC50(0.053±0.001)μg/ml in gemcitabine groups. When Ad-PUMA or gemecitabine was injected as a single agent it resultd in reduction in 62.75(P<0.01 vs control) or 42.3% (p<0.05 vs control)tumor growth ,however, the combination of Ad-PUMA with gemcitabine reduced tumor volume by more than 82%(P<0.001 vs control)on day 21.
Conclusion PUMA significantly enhances chemosensitivity of AsPC-1 to gemcitabine, and PUMA and gemcitabine exert anadditive anti-tumor effect in vivo.
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