新型乙型肝炎病毒载体的构建及初步应用研究
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摘要
乙型肝炎病毒(HBV)载体是近几年新发展的一种病毒载体,不仅具有天然嗜肝特异性,而且具备改造为肝靶向性基因治疗载体的基本条件。主要包括:在肝细胞内持续复制、反复感染,病毒本身对细胞没有明显的细胞毒性;能够携带外源基因并被包装成病毒颗粒;能够介导外源基因的转移和表达;但由于基因结构复杂,目前改造的HBV载体均存在载容量小、复制包装效率低及安全性差等缺点,而且HBV载体制备困难,感染效率低,为解决目前存在的困难,有必要使其与其他病毒载体联合应用。如与腺病毒载体、慢病毒载体、杆状病毒载体或逆转录病毒载体联合应用,实现高载容量、高转染效率、高复制能力,充分发挥HBV载体嗜肝导向性,可以利用体内野生型HBV为之提供包装,在体内形成具有抗HBV作用的HBV样颗粒,或携带治疗性目的基因,进一步感染其他肝细胞,实现放大效应。因此,通过病毒载体之间的重组,拓宽了HBV载体应用领域,不仅有望用于抗HBV基因治疗,也可以用于制备DNA疫苗、筛选抗病毒药物或通过携带外源性标志基因用于HBV分子生物学研究等。本研究通过构建新型的HBV载体表达不同的目的基因,构建了持续分泌表达BsdR重组HBV的的细胞系,用于HBV的分子生物学研究或筛选HBV易感细胞系;或与腺病毒载体联合构建了表达MMP8等目的基因的质粒,用于抗肝硬化的基因治疗,为HBV的分子生物学研究和肝硬化的生物治疗等开辟了新途径。本研究共分3部分完成。
第一部分新型HBV载体的构建
目的应用分子生物学技术改造HBV载体,阻断其结构基因的表达,提高HBV载体作为基因治疗载体的安全性,使其更适用于作为基因治疗载体。
方法:
1 pCH-M5-GFP质粒的构建通过改变pCH-S-GFP的起始密码子或添加终止密码子,删除了HBV质粒中Core、HBV-DNAP、pre-S1、pre-S2、S and X等结构蛋白的表达,构建pCH-M5-GFP质粒。
2 pCH-S-hRLuc和pCH-M5-hRLuc的构建在pCH-S-GFP和pCH-M5-GFP质粒S基因区,以hRLuc替换GFP,构建pCH-S-hRLuc和pCH-M5-hRLuc两个质粒。
3外源基因的表达能力的检测HBV载体经基因改造后,通过质粒共转染肝细胞,应用荧光显微镜观察绿色荧光蛋白的表达,应用荧光素酶检测试剂盒检测海肾荧光素酶的表达水平,鉴定新构建质粒pCH-M5-GFP、pCH-S-hrLuc和pCH-M5-hrLuc的外源基因的表达能力。
4重组HBV复制能力的检测。与辅助质粒共转染后,提取细胞裂解液中的HBVDNA,Sorthern blot技术检测重组HBV复制能力。
5重组HBV感染树鼩肝细胞能力的检测。原代培养树鼩肝细胞,感染浓缩的重组HBV颗粒1W后,观察细胞内GFP的表达水平。
结果:
1成功构建了不能表达HBV结构基因的新型HBV载体。
2新型HBV载体外源基因的表达水平基因突变后表达GFP的质粒转染肝癌细胞系后,GFP表达水平明显强于原始质粒。
3通过southern blot检测,在细胞裂解液中能够形成HBV复制中间体。
4重组HBV感染树鼩肝细胞1W后,能够观察到GFP。
结论:HBV载体通过变更起始密码子和终止密码子后,阻断结构基因表达,在S基因区插入报告基因GFP和hrLUC,构建了新质粒,其外源基因的表达水平高于对照原始载体质粒,能够形成HBV DNA复制中间体,而且形成的重组HBV能够感染树鼩肝细胞。
第二部分新型HBV载体应用一:持续分泌表达BsdR的重组HBV细胞系的构建
目的:
构建一个持续分泌重组HBV的细胞系,该重组HBV表达杀稻瘟菌素抗性基因(BsdR)。
方法:
1构建具有G418抗性的无包装信号的HBV辅助质粒pcDNA3.1-CH3142与表达BsdR的HBV载体质粒。
2上述2个质粒共转染HepG2细胞,经G418和杀稻瘟菌素共筛选,获得阳性细胞克隆。
3经过ELISA和斑点杂交技术筛选出高分泌重组HBV-Bsd颗粒的细胞系。
4 G418反复加压培养,Southern blot和Native Western blot技术,再次筛选,获得高表达Bsd的重组HBV的细胞系,并通过PCR技术检测重组HBV的表达水平。
5应用PCR技术,对野生型HBV和重组HBV进行鉴定,证实所构建细胞系无野生型HBV形成。
6应用免疫电镜技术观察到完整的有囊膜包被的重组HBV颗粒。
结果:
1转染后,经G418筛选后,获得50余个细胞克隆。
2挑选其中36个进行扩增,经同位素标记探针斑点杂交法检测细胞上清液中HBV DNA,选取9个表达量较高者。
3继续传代培养15代(约3个月),检测其形成疏松环状DNA的能力,最终选定HBV-Bsd25,HBV-Bsd7,HBV-Bsd27三个细胞株。
4细胞培养上清液中HBV DNA定量分别为4.1×106、3.6×106、1.2×106copies/mL。
5未检测到野生型HBV的产生。
6 HBV-Bsd25细胞培养上清液经PEG8000浓缩后进行氯化铯密度梯度离心,分段进行Southern Blot及Western Blot检测,观察到完整有囊膜包被的重组HBV颗粒形成。
7电镜观察有完整的HBV颗粒。
结论:成功构建稳定表达BsdR重组HBV的细胞系,实现了大量制备重组HBV载体,有助于HBV载体用于HBV易感细胞系的筛选。
第三部分新型HBV载体质粒应用二:腺病毒-HBV嵌合载体表达胶原酶II抗肝硬化研究
一、腺病毒-HBV嵌合载体的构建与表达
目的构建表达MMP8、tMMP8、RFP2的HBV载体,再利用复制缺损型腺病毒载体进行包装,构建Ad-CH-tMMP8、Ad-C-MMP8、Ad-CH-RFP2三个质粒。观察细胞内tMMP8 mRNA和RFP的表达情况,证明目的基因的表达能力,及其启动子的嗜肝特异性。
方法:
1应用PCR技术和酶切重组技术构建表达MMP8、tMMP8、RFP2的HBV载体,经过酶切鉴定和测序鉴定正确后,再利用复制缺损型腺病毒载体进行包装,构建Ad-CH-tMMP8、Ad-C-MMP8、Ad-CH-RFP2三个质粒。
2 PCR技术证实重组腺病毒载体正确性,获得理想基因片段。
3正确后,进行大量提取,纯化,浓度测定,感染293细胞,进行空斑形成能力的检测。
4肝细胞感染重组腺病毒后,观察细胞内MMP8及tMMP8 mRNA的表达情况,应用荧光显微镜RFP的表达情况.
5 HBV载体在血管上皮细胞(ECV304)中低表达:以CMV启动子及HBVS启动子驱动的GFP质粒,转染血管上皮细胞及肝癌细胞,分别观察其GFP的表达。
结果:
1成功构建了表达不同目的基因的腺病毒-HBV嵌合载体质粒。
2携带MMP8和tMMP8的重组腺病毒感染肝细胞后表达tMMP8。携带RFP2的重组腺病毒感染肝细胞后表达红色荧光。
3不同启动子驱动的GFP质粒转染不同细胞后,在HepG2和ECV304的表达水平存在差异。
结论:表达不同目的基因的腺病毒载体-HBV嵌合载体能够被成功构建。该载体转染肝癌细胞后,能够成功表达各种目的基因;重组腺病毒载体携带MMP8基因的质粒感染HepG2后,其细胞中存在MMP8 mRNA的表达,应用荧光显微镜可以观察到红色荧光蛋白;HBV载体s启动子驱使的GFP在ECV304中低表达,在HepG2呈现高表达。而CMV启动子驱使的GFP在ECV304和HepG2均呈现高表达,提示S启动子的载体质粒对肝癌细胞系有一定特异性。
二、硫代乙酰胺诱导的大鼠肝硬化模型的实验研究
目的观察TAA制备大鼠肝硬化模型前后,肝脏组织学及血清学指标的变化情况,HGF/C-met系统在肝硬化发生和转归过程中的表达水平,为腺病病毒-HBV前忽而载体表达胶原酶II在肝硬化的实验研究奠定基础。
方法: 42只雄性Wistar大鼠应用硫代乙酰胺饮用水喂养,制备肝硬化模型。模型制备成功后,随机分为7组,每组6只。每隔2周处死一组大鼠,留取血清检测肝功能,并检测肝组织中羟脯氨酸含量,进行HE和天狼猩红染色,同时RT-PCR技术检测HGF及其受体c-Met,另以正常大鼠6只为对照。
结果:HGF/c-Met mRNA表达显著高于正常对照组,随着停止应用TAA时间的延长,肝功能、组织学改变、羟脯氨酸含量及HGF/c-Met的mRNA表达水平均逐渐恢复。
结论:应用TAA20周成功诱导了肝硬化模型;停止TAA后,肝组织学、肝功能和肝脏的羟脯氨酸均有不同程度的自愈趋势。肝脏组织学的好转晚于肝功能的变化。在肝组织自愈的的同时,HGF及其受体c-Met的表达水平均有不同程度的下降趋势,但各组之间无统计学差异。
三、腺病毒-HBV嵌合载体表达胶原酶Ⅱ治疗肝硬化的实验研究
目的通过利用腺病毒-HBV嵌合载体,表达胶原酶Ⅱ,治疗肝硬化大鼠模型,探讨其能否有效地降解Ⅰ型胶原,促进肝细胞再生,改善肝功能,逆转肝硬化。
方法:
1模型制备应用0.3%硫代乙酰胺饮用水诱导的方法,制备大鼠肝硬化模型。
2分组给药120只肝硬化模型大鼠随机分四组,每组30只,分别给予Ad-CH-tMMP8、Ad-C-MMP8、Ad-CH-RFP2三种重组腺病毒尾静脉注射,以1.5×1011VP/Kg剂量,一次给药。另外留取30只大鼠作为正常对照组,给予正常应用水喂养。
3标本留取分别于2W、4W、8W后颈椎脱髓法处死动物。分离血清,-20℃保存;留取肝组织,少部分用10%的福尔马林固定,进行组织病理学检查,余者无菌条件下保存在液氮中。
4指标检测全自动生化分析仪检测肝功能、放射免疫法检测肝硬化指标。胃酸酶解法检测肝组织羟脯氨酸含量。HE、天狼星红染色、免疫组化的方法观察肝脏组织病理学改变,并进行Knodel评分和一型胶原所占面积百分比进行半定量分析。RT-PCR方法检测肝组织内MMP8、HGF、c-Met和GAPDH。
5统计分析应用SAS统计分析软件,进行统计处理。
结果:
1 Ad-HBV嵌合载体对肝硬化大鼠模型MMP8 mRNA表达水平的影响治疗组大鼠肝脏能够表达MMP8,但对照组没有检测到MMP8 mRNA表达,而且随着停药时间的延长,其表达水平呈下降趋势,直到第12W仍有少量表达。
2 Ad-HBV嵌合体对肝硬化大鼠模型肝功能的影响在第2W、第4W时,与两个对照组相比,各治疗组ALT均有明显下降,具有统计学意义(P<0.05)。第8周,治疗组肝功能接近正常组。
3 Ad-HBV嵌合体对肝硬化大鼠模型肝纤维化指标的影响与两个对照组相比,各治疗组HA和LN均有明显下降,具有统计学意义(P<0.05)。在治疗第8W时,治疗组HA和LN接近正常组。
4 Ad-HBV嵌合体对大鼠肝硬化模型肝组织学的影响与对照组相比,治疗组病理学改善较快,炎症细胞浸润及纤维组织增生明显减轻,肝小叶结构破坏减轻,有肝细胞再生现象。
5 Ad-HBV嵌合体对大鼠肝硬化模型肝组织羟脯氨酸含量的影响羟脯氨酸是胶原和弹性蛋白水解后产生的的一种氨基酸,约占胶原重量14%,其他蛋白质中罕见。羟脯氨酸的含量也随着肝硬化的严重程度而改变,在应用腺病毒治疗2W时,肝组织内羟脯氨酸含量明显低于对照组。
6 Ad-HBV嵌合体在大鼠肝硬化模型HGF/c-Met mRNA的表达与两个对照组相比,各治疗组HGF/cMet mRNA均有明显升高,具有统计学意义(P<0.05)。第8周,治疗组肝组织HGF/c-Met mRNA接近正常组。
结论:
应用重组腺病毒治疗大鼠肝硬化模型后,肝功能好转,肝组织学及肝组织内的羟脯胺酸明显改善,肝组织表达MMP8,能够有效地降解Ⅰ型胶原,肝硬化减轻,肝细胞再生。HGF/c-Met mRNA表达水平增加。这一创新,有望把MMP-8基因治疗肝硬化带入临床应用,开辟肝硬化软肝治疗新途径。
Hepatitis B virus (HBV) vector is a new type of viral vector found in recent years, which is different from viral vector commonly used such as adenovirus, adeno-associated virus, retrovirus, lentivirus, etc. It has great advantages over other vectors for its specifically targeting to the hepatocytes and its basic condition for reconstructing into hepa-target gene therapy vector. The advantages of the HBV vector include self-replicating continuously in liver cells, infecting repeatedly and no conspicuous cytotoxicity, packing viral particle, expressing, transferring and carrying exogenous genes. The disadvantage of HBV vector is that the gene structure is too complicated to content enough exogenous genes. It possesses low copy packaging efficiency and low security, low efficiency of infection. In addition, it is difficult for preparation. In order to solve these difficulties, it is very necessary to associate with other viral vectors such as adenovirus, adeno-associated virus, retrovirus, lentivirus, etc. Only by doing so, can high carry capability, transfection efficiency and replication be realized. As HBV vector is hepatotropic, it is thoroughly educed, enveloped by utilizing wild type HBV in the human body and forms reconstruction HBV particles with anti-HBV effects, or carrying therapeutic target genes and further infects other liver cells to realize amplification effectiveness. Therefore, by reconstruction among the viral vectors, the application domain of HBV vector is widened deeply. Therefore it may be used not only for anti-HBV gene therapy, but also for HBV molecular biological research by expressing special exogenous gene.
On this study a stable cell line with permanent secretion of recombinant hepatitis B virus (HBV) was constructed through the construction of HBV vector with expression of different target gene, which is useful for molecular biological study or screening of HBV susceptible cell line. It can also be used together with adenovirus to construct target gene plasmids which express MMP8 and can be used for anti-cirrhosis gene therapy. Therefore, this study developed a new way for molecular biological research on HBV and biological treatment of cirrhosis. This study includes three parts.
Part one Construction of a new type HBV Vector
objective To explore an effective ways constructing and making HBV vector plasimids, which cannot express any structural genes ,by molecular biology technique. The new type HBV vector may be more security and applicable as a gene therapy vector.
Method
1 The original genes (Core, Polymerase, pre-S1, pre-S2, S and X) of HBV vector (pCH-S-GFP) were deleted by altering the start codon or adding stop codons, pCH-M5-GFP was successfuly constructed.
2 Deleting the GFP genes of pCH-S-GFP and pCH-M5-GFP, then inserting the hRLuc gene into the S region, pCH-S-hRLuc and pCH-M5-hRLuc were successfuly constructed.
3. To determine exogenous gene expression levels, after hepatocyte was cotransfected by the new plasimids and pCH3142, GFP and hRLuc were observed by fluorescence microscope or Luminometer.
4 To observed the ability of recombinant HBV replication, HBV DNA in cell lysate was extracted and southern blot was done to detect HBV replication intermediates, after plasmid cotransfection.
5 To detect the infectious capacity of the recombinant HBV on tree shrews hepatocyte tree shrews hepatocytes were infected by concentration and purification of reHBV particles from cell supernatant. The expression level of GFP would be observed after 1W.
Results
1 The three HBV vector plasmids were successfully constructed by gene recombination technology.
2 After the cells cotransfected by the mutation plasmid, expression level of GFP and hRLuc was stronger than the original plasmid.
3 Cotransfected by the mutation plasmid and pCH3142, the formation of HBV replication intermediates can be observed in Cell lysate by southern blot.
4 Expresstion of GFP can be observed after reHBV infected tree shrews hepatocytes.
Conclusion After the start codon and terminal codons of the HBV plasmids were mutated, HBV structure protein can not be expressed;Some report genes can be inserted into the S region of the new HBV vector plasmids, and stronger expression than the original plasmid;ReHBV can infect tree shrews hepatocytes.
Part two The first aspect application of the new type HBV vector---Stable cell line for secretion of replication-defective hepatitis B virus vector expressing blasticidin resistant gene
Objective:To construct a stable cell line with permanent secretion of recombinant hepatitis B virus(HBV)vector which express blasticidin resistant gene.
Method
1 Construction of HBV helper plasmid pcDNA3.1-CH3142 with G418-resistent non packing signal and HBV vector plasmids which express blasticidin resistant gene.
2 HepG2 cells were cotransfected with both the HBV vector expressing blasticidin resistant gene and the helper plasmids with G418-resistant gene. Cell clones were selected by the addition of both blasticidin and G418. Positive cell clones were gained.
3 Screening cell clones with high secretion of the HBV-Bsd recombinant hepatitis B virus particles by ELISA and dot blot hybridization
4 After culturing by the addition of both G418 and Bsd, regaining the higher level recombinant HBV cell line for secretion of replication-defective hepatitis B virus by Southernblot and Native Westernblot technology, and test the expression level of recombinant HBV through PCR
5 Application of PCR technology, wild-type hepatitis B virus and recombinant hepatitis B virus were identified and confirmed wild-type hepatitis B virus can not be formed in the new cell lines.
6 The complete recombinated HBV particles were tested by immune electron microscopy.
Result
1 Over 50 cell clones were formed through G418 screening after transfection.
2 36 cell clones were picked and expanded. HBV DNA in the supernatant was tested by dot blot hybridization with isotope labeled probe. Afterward the best 9 clones were selected
3 After maintaining for 15 generations (about 3 months), HBV DNA was proved by southern blot with isotope labeled probe. Confirmed the formation of RC DNA, According to the results, 3 cell lines of HBV-Bsd25,HBV-Bsd7, HBV-Bsd27 were chosen.
4 The quantity of HBV DNA of the three best cell lines (HBV-Bsd25, HBV-Bsd7, HBV-Bsd27) were 4.1*106, 3.6*106 and 1.2*106 copies/mL respectively.
5 No wild type HBV was detected.
6 Cesium chloride density gradient analysis in HBV-Bsd25 cell lines was done with the concentrated supernatant HBV virion, and enveloped recombinant HBV were proved by southern blot and HBsAg western blot.
7 Complete HBV particles were observed by electronic microscope.
Conclusion By the methods of HBV vector and HBV helper plasmids cotransfection, we could obtain the stable cell lines with permanent secretion of HBV virions and realized large preparation of recombinant HBV virions. This will contribute to the use of HBV vector for gene therapy. The recombinant HBV secretion stable cell line will also useful for the ongoing selection of HBV susceptible cell line.
Part three The second aspect application of the new type HBV vector plasmids---
An experimental study on rat liver cirrhosis model administrated with Adenovirus-HBV chimeric vector expressing collagenase II
No. 1 : construction and expression of adenovirus chimeric HBV vector in vitro
Objective To construct 3 adenovirus chimeric HBV vector plasmids which expressing different purpose gene, then observe the expressing levels of tMMP8 mRNA and RFP in vitro. To detect the expression of GFP in ECV304 and HepG2 with a CMV promoter and the HBV S promoter-driven GFP plasmids.
Method
1 Three new plasmids expressing purpose gene were successfully constructed.
2 To confirm the correctness of recombined adenovirus vector by PCR technology, the ideal gene fragments were obtained.
3 After three new correct plasmids were amplificated, extracted and purified, concentration determination and plaque-forming ability were detected.
4 After the three recombinant adenovirus infected HepaG2, RFP was detected by fluorescence microscopy, tMMP mRNA detected by RT-PCR technique.
2 After ECV304 and HepG2 were transfected with the plasmids of CMV promoter and the HBV S promoter-driven GFP, the expression of GFP was detected with fluorescence microscopy.
Result
1 HBV-adenovirus chimeric vector carrying different purpose genes were successfully constructed.
2 After recombinant adenovirus carrying MMP8 and tMMP8 infected hepatocytes, MMP8 and tMMP8 mRNA expression can be observed, recombinant adenovirus carrying RFP2 in hepatocytes can express RFP.
3 Defferent promoter-driven GFP plasmids transfected defferent cell lines, the level of GFP expression is defferent.
Conclusion Adenovirus vector can packaged HBV vector and expressing different purpose genes and formation HBV-adenovirus chimeric vector. After transfeced HepG2 cell lines, recombinated adenovirus vector can be successful expressing different purpose gene. HBV carrier s promoter-driven GFP in the vascular epithelial cells (ECV304) is low expression, in liver cell lines showed high expression. And CMV promoter-driven GFP in the vascular epithelial cells (ECV304) and liver cell lines showed high expression, suggesting that S promoter plasmid posseses hepatotropic.
No.2: An experimental study on rat liver cirrhosis model induced by TAA
Objective To explore the level of HGF/C-met, Liver histology, liver function and liver hydroxyproline on TAA induced rat liver cirrhosis model.
Method 42 male Wistar rats were fed with thioacetamide drinking water, preparation of liver fibrosis model. After the success of the model preparation, the rats were randomly divided into 7 groups of 6. Every two weeks killed a group of rats, collecting serum for liver function testing and detection of liver tissue hydroxyproline content, for HE and Sirius Red staining, and RT-PCR detected HGF and its receptor c-Met, and the other six normal rats were set as control group.
Result HGF/ c-Met mRNA expression was significantly higher than the normal control group. With the time to stop the extension of the application of TAA, liver function, histological changes, hydroxyproline content and HGF / c-Met mRNA expression level were returned to normal and a positive correlation was observed.
Conclusion:Rat liver cirrhosis models can be induced successfuly by TAA administrated for 20 weeks. After ceasing TAA, Liver histology, liver function and liver hydroxyproline have varying degrees of self-healing trend. Self-healing in the liver tissue at the same time, HGF and its receptor c-Met expression levels in varying degrees downward trend, however, no significant difference between groups. The occurrence of liver injury, an increase in HGF / c-Met expression level may be adopted to promote liver regeneration and reverse liver fibrosis
No.3: An experimental study on rat liver cirrhosis model administrated with Adenovirus-HBV chimeric vector expressing collagenase II
Objective Neutrophil collagenase (MMP-8) can degrade typeⅠcollagen specifically. Introduction of MMP-8 gene into liver cells could be an advantageous tool to degrade collagen. In this study, the aim is to explore the effectiveness of the typeⅠcollagen degradation, and the promotion of liver cell regeneration and improvement of liver function, to reduce liver fibrosis through treating cirrhosis rat model in vivo with Adenovirus-HBV chimeric vector expressing collagenase II
Method
1 Rat liver fibrosis model was prepared by 0.3% thioacetamide-induced drinking water.
2 Randomized and divided into groups and drug delivery 120 liver fibrosis model rats were randomly divided into four groups, were given Ad-CH-tMMP8, Ad-C-MMP8, Ad-CH-RFP2, tail vein injection of recombinant adenovirus, 1.5×1011 viral particles/Kg dose, one administration. Selecting another 30 rats as normal control group, administrated the normal water.
3 Ten rats in each group were sacrificed in 2W, 4W, 8W, respectively, and separate the serum, -20℃preservation. part of liver tissue were put in 10% of the formalin-fixed for histopathological examination, the remainder of the liver tissue were stored in liquid nitrogen.
4 Testing liver function by automatic biochemical analyzer, Hydroxyproline in liver tissue by acid hydrolysis method, the liver histopathological changes by HE, sirius red staining and immunohistochemical method, MMP8, HGF and c-Met and GAPDH mRNA by RT-PCR.
5 Statistical analysis. Statistical analysis was conducted by SAS statistical analysis software.
Result:
1 The expression level of MMP8 mRNA. MMP8 mRNA in the treatment group could be expressed in rat liver, but no MMP8 mRNA expression in the control group. The expression level of MMP8 mRNA was downward trend, with the extension of treatment time.
2 Liver function. As compared with two control groups, ALT in the two treatment groups were significantly decreased at 2 W, 4W and 8W(P <0.05), ALT in the two treatment groups was near to the normal group at 8W.
3 Indicators of liver fibrosis. HA and LN in the two treatment groups were significantly decreased at 2 W, 4W and 8W (P <0.05), HA and LN in the two treatment groups was near normal group at 8W.
4 Liver histology pathology Compared with the control group,pathology in the treatment group improve significantly,Inflammatory cell infiltration and the proliferation of tissue were significantly reduced. Hepatic lobules is damaged to a lesser extent. There is the phenomenon of liver regeneration
5 The quantity of Hydroxyproline. Hydroxyproline is an amino acid derived from collagen and elastin hydrolysis, accounting for about 14% of the weight of collagen. It is rare in other proteins. The quantity of hydroxyproline is varied with the severity of liver cirrhosis. At 2W application of the treatment of adenovirus, in the liver tissue, the hydroxyproline quantity was significantly lower than that of the control group
6 The expression level of HGF/cMet mRNA. HGF/cMet mRNA in the two treatment groups were significantly increased at 2 W, 4W and 8W(P <0.05) compared with control groups.
Conclusion:After application of recombinant adenovirus, liver tissue can express MMP8. The latter can effectively degrade collagen typeⅠ, reduce liver fibrosis, liver cells regenerate. The expression level of HGF / cMet mRNA was increased. This innovation is expected that MMP-8 gene therapy is brought into the clinical application of liver cirrhosis, it is a new strategy of treatment for reversing liver cirrhosis.
第一部分新型HBV载体的构建
目的应用分子生物学技术改造HBV载体,阻断其结构基因的表达,提高HBV载体作为基因治疗载体的安全性,使其更适用于作为基因治疗载体。
方法:
1 pCH-M5-GFP质粒的构建通过改变pCH-S-GFP的起始密码子或添加终止密码子,删除了HBV质粒中Core、HBV-DNAP、pre-S1、pre-S2、S and X等结构蛋白的表达,构建pCH-M5-GFP质粒。
2 pCH-S-hRLuc和pCH-M5-hRLuc的构建在pCH-S-GFP和pCH-M5-GFP质粒S基因区,以hRLuc替换GFP,构建pCH-S-hRLuc和pCH-M5-hRLuc两个质粒。
3外源基因的表达能力的检测HBV载体经基因改造后,通过质粒共转染肝细胞,应用荧光显微镜观察绿色荧光蛋白的表达,应用荧光素酶检测试剂盒检测海肾荧光素酶的表达水平,鉴定新构建质粒pCH-M5-GFP、pCH-S-hrLuc和pCH-M5-hrLuc的外源基因的表达能力。
4重组HBV复制能力的检测。与辅助质粒共转染后,提取细胞裂解液中的HBVDNA,Sorthern blot技术检测重组HBV复制能力。
5重组HBV感染树鼩肝细胞能力的检测。原代培养树鼩肝细胞,感染浓缩的重组HBV颗粒1W后,观察细胞内GFP的表达水平。
结果:
1成功构建了不能表达HBV结构基因的新型HBV载体。
2新型HBV载体外源基因的表达水平基因突变后表达GFP的质粒转染肝癌细胞系后,GFP表达水平明显强于原始质粒。
3通过southern blot检测,在细胞裂解液中能够形成HBV复制中间体。
4重组HBV感染树鼩肝细胞1W后,能够观察到GFP。
结论:HBV载体通过变更起始密码子和终止密码子后,阻断结构基因表达,在S基因区插入报告基因GFP和hrLUC,构建了新质粒,其外源基因的表达水平高于对照原始载体质粒,能够形成HBV DNA复制中间体,而且形成的重组HBV能够感染树鼩肝细胞。
第二部分新型HBV载体应用一:持续分泌表达BsdR的重组HBV细胞系的构建
目的:
构建一个持续分泌重组HBV的细胞系,该重组HBV表达杀稻瘟菌素抗性基因(BsdR)。
方法:
1构建具有G418抗性的无包装信号的HBV辅助质粒pcDNA3.1-CH3142与表达BsdR的HBV载体质粒。
2上述2个质粒共转染HepG2细胞,经G418和杀稻瘟菌素共筛选,获得阳性细胞克隆。
3经过ELISA和斑点杂交技术筛选出高分泌重组HBV-Bsd颗粒的细胞系。
4 G418反复加压培养,Southern blot和Native Western blot技术,再次筛选,获得高表达Bsd的重组HBV的细胞系,并通过PCR技术检测重组HBV的表达水平。
5应用PCR技术,对野生型HBV和重组HBV进行鉴定,证实所构建细胞系无野生型HBV形成。
6应用免疫电镜技术观察到完整的有囊膜包被的重组HBV颗粒。
结果:
1转染后,经G418筛选后,获得50余个细胞克隆。
2挑选其中36个进行扩增,经同位素标记探针斑点杂交法检测细胞上清液中HBV DNA,选取9个表达量较高者。
3继续传代培养15代(约3个月),检测其形成疏松环状DNA的能力,最终选定HBV-Bsd25,HBV-Bsd7,HBV-Bsd27三个细胞株。
4细胞培养上清液中HBV DNA定量分别为4.1×106、3.6×106、1.2×106copies/mL。
5未检测到野生型HBV的产生。
6 HBV-Bsd25细胞培养上清液经PEG8000浓缩后进行氯化铯密度梯度离心,分段进行Southern Blot及Western Blot检测,观察到完整有囊膜包被的重组HBV颗粒形成。
7电镜观察有完整的HBV颗粒。
结论:成功构建稳定表达BsdR重组HBV的细胞系,实现了大量制备重组HBV载体,有助于HBV载体用于HBV易感细胞系的筛选。
第三部分新型HBV载体质粒应用二:腺病毒-HBV嵌合载体表达胶原酶II抗肝硬化研究
一、腺病毒-HBV嵌合载体的构建与表达
目的构建表达MMP8、tMMP8、RFP2的HBV载体,再利用复制缺损型腺病毒载体进行包装,构建Ad-CH-tMMP8、Ad-C-MMP8、Ad-CH-RFP2三个质粒。观察细胞内tMMP8 mRNA和RFP的表达情况,证明目的基因的表达能力,及其启动子的嗜肝特异性。
方法:
1应用PCR技术和酶切重组技术构建表达MMP8、tMMP8、RFP2的HBV载体,经过酶切鉴定和测序鉴定正确后,再利用复制缺损型腺病毒载体进行包装,构建Ad-CH-tMMP8、Ad-C-MMP8、Ad-CH-RFP2三个质粒。
2 PCR技术证实重组腺病毒载体正确性,获得理想基因片段。
3正确后,进行大量提取,纯化,浓度测定,感染293细胞,进行空斑形成能力的检测。
4肝细胞感染重组腺病毒后,观察细胞内MMP8及tMMP8 mRNA的表达情况,应用荧光显微镜RFP的表达情况.
5 HBV载体在血管上皮细胞(ECV304)中低表达:以CMV启动子及HBVS启动子驱动的GFP质粒,转染血管上皮细胞及肝癌细胞,分别观察其GFP的表达。
结果:
1成功构建了表达不同目的基因的腺病毒-HBV嵌合载体质粒。
2携带MMP8和tMMP8的重组腺病毒感染肝细胞后表达tMMP8。携带RFP2的重组腺病毒感染肝细胞后表达红色荧光。
3不同启动子驱动的GFP质粒转染不同细胞后,在HepG2和ECV304的表达水平存在差异。
结论:表达不同目的基因的腺病毒载体-HBV嵌合载体能够被成功构建。该载体转染肝癌细胞后,能够成功表达各种目的基因;重组腺病毒载体携带MMP8基因的质粒感染HepG2后,其细胞中存在MMP8 mRNA的表达,应用荧光显微镜可以观察到红色荧光蛋白;HBV载体s启动子驱使的GFP在ECV304中低表达,在HepG2呈现高表达。而CMV启动子驱使的GFP在ECV304和HepG2均呈现高表达,提示S启动子的载体质粒对肝癌细胞系有一定特异性。
二、硫代乙酰胺诱导的大鼠肝硬化模型的实验研究
目的观察TAA制备大鼠肝硬化模型前后,肝脏组织学及血清学指标的变化情况,HGF/C-met系统在肝硬化发生和转归过程中的表达水平,为腺病病毒-HBV前忽而载体表达胶原酶II在肝硬化的实验研究奠定基础。
方法: 42只雄性Wistar大鼠应用硫代乙酰胺饮用水喂养,制备肝硬化模型。模型制备成功后,随机分为7组,每组6只。每隔2周处死一组大鼠,留取血清检测肝功能,并检测肝组织中羟脯氨酸含量,进行HE和天狼猩红染色,同时RT-PCR技术检测HGF及其受体c-Met,另以正常大鼠6只为对照。
结果:HGF/c-Met mRNA表达显著高于正常对照组,随着停止应用TAA时间的延长,肝功能、组织学改变、羟脯氨酸含量及HGF/c-Met的mRNA表达水平均逐渐恢复。
结论:应用TAA20周成功诱导了肝硬化模型;停止TAA后,肝组织学、肝功能和肝脏的羟脯氨酸均有不同程度的自愈趋势。肝脏组织学的好转晚于肝功能的变化。在肝组织自愈的的同时,HGF及其受体c-Met的表达水平均有不同程度的下降趋势,但各组之间无统计学差异。
三、腺病毒-HBV嵌合载体表达胶原酶Ⅱ治疗肝硬化的实验研究
目的通过利用腺病毒-HBV嵌合载体,表达胶原酶Ⅱ,治疗肝硬化大鼠模型,探讨其能否有效地降解Ⅰ型胶原,促进肝细胞再生,改善肝功能,逆转肝硬化。
方法:
1模型制备应用0.3%硫代乙酰胺饮用水诱导的方法,制备大鼠肝硬化模型。
2分组给药120只肝硬化模型大鼠随机分四组,每组30只,分别给予Ad-CH-tMMP8、Ad-C-MMP8、Ad-CH-RFP2三种重组腺病毒尾静脉注射,以1.5×1011VP/Kg剂量,一次给药。另外留取30只大鼠作为正常对照组,给予正常应用水喂养。
3标本留取分别于2W、4W、8W后颈椎脱髓法处死动物。分离血清,-20℃保存;留取肝组织,少部分用10%的福尔马林固定,进行组织病理学检查,余者无菌条件下保存在液氮中。
4指标检测全自动生化分析仪检测肝功能、放射免疫法检测肝硬化指标。胃酸酶解法检测肝组织羟脯氨酸含量。HE、天狼星红染色、免疫组化的方法观察肝脏组织病理学改变,并进行Knodel评分和一型胶原所占面积百分比进行半定量分析。RT-PCR方法检测肝组织内MMP8、HGF、c-Met和GAPDH。
5统计分析应用SAS统计分析软件,进行统计处理。
结果:
1 Ad-HBV嵌合载体对肝硬化大鼠模型MMP8 mRNA表达水平的影响治疗组大鼠肝脏能够表达MMP8,但对照组没有检测到MMP8 mRNA表达,而且随着停药时间的延长,其表达水平呈下降趋势,直到第12W仍有少量表达。
2 Ad-HBV嵌合体对肝硬化大鼠模型肝功能的影响在第2W、第4W时,与两个对照组相比,各治疗组ALT均有明显下降,具有统计学意义(P<0.05)。第8周,治疗组肝功能接近正常组。
3 Ad-HBV嵌合体对肝硬化大鼠模型肝纤维化指标的影响与两个对照组相比,各治疗组HA和LN均有明显下降,具有统计学意义(P<0.05)。在治疗第8W时,治疗组HA和LN接近正常组。
4 Ad-HBV嵌合体对大鼠肝硬化模型肝组织学的影响与对照组相比,治疗组病理学改善较快,炎症细胞浸润及纤维组织增生明显减轻,肝小叶结构破坏减轻,有肝细胞再生现象。
5 Ad-HBV嵌合体对大鼠肝硬化模型肝组织羟脯氨酸含量的影响羟脯氨酸是胶原和弹性蛋白水解后产生的的一种氨基酸,约占胶原重量14%,其他蛋白质中罕见。羟脯氨酸的含量也随着肝硬化的严重程度而改变,在应用腺病毒治疗2W时,肝组织内羟脯氨酸含量明显低于对照组。
6 Ad-HBV嵌合体在大鼠肝硬化模型HGF/c-Met mRNA的表达与两个对照组相比,各治疗组HGF/cMet mRNA均有明显升高,具有统计学意义(P<0.05)。第8周,治疗组肝组织HGF/c-Met mRNA接近正常组。
结论:
应用重组腺病毒治疗大鼠肝硬化模型后,肝功能好转,肝组织学及肝组织内的羟脯胺酸明显改善,肝组织表达MMP8,能够有效地降解Ⅰ型胶原,肝硬化减轻,肝细胞再生。HGF/c-Met mRNA表达水平增加。这一创新,有望把MMP-8基因治疗肝硬化带入临床应用,开辟肝硬化软肝治疗新途径。
Hepatitis B virus (HBV) vector is a new type of viral vector found in recent years, which is different from viral vector commonly used such as adenovirus, adeno-associated virus, retrovirus, lentivirus, etc. It has great advantages over other vectors for its specifically targeting to the hepatocytes and its basic condition for reconstructing into hepa-target gene therapy vector. The advantages of the HBV vector include self-replicating continuously in liver cells, infecting repeatedly and no conspicuous cytotoxicity, packing viral particle, expressing, transferring and carrying exogenous genes. The disadvantage of HBV vector is that the gene structure is too complicated to content enough exogenous genes. It possesses low copy packaging efficiency and low security, low efficiency of infection. In addition, it is difficult for preparation. In order to solve these difficulties, it is very necessary to associate with other viral vectors such as adenovirus, adeno-associated virus, retrovirus, lentivirus, etc. Only by doing so, can high carry capability, transfection efficiency and replication be realized. As HBV vector is hepatotropic, it is thoroughly educed, enveloped by utilizing wild type HBV in the human body and forms reconstruction HBV particles with anti-HBV effects, or carrying therapeutic target genes and further infects other liver cells to realize amplification effectiveness. Therefore, by reconstruction among the viral vectors, the application domain of HBV vector is widened deeply. Therefore it may be used not only for anti-HBV gene therapy, but also for HBV molecular biological research by expressing special exogenous gene.
On this study a stable cell line with permanent secretion of recombinant hepatitis B virus (HBV) was constructed through the construction of HBV vector with expression of different target gene, which is useful for molecular biological study or screening of HBV susceptible cell line. It can also be used together with adenovirus to construct target gene plasmids which express MMP8 and can be used for anti-cirrhosis gene therapy. Therefore, this study developed a new way for molecular biological research on HBV and biological treatment of cirrhosis. This study includes three parts.
Part one Construction of a new type HBV Vector
objective To explore an effective ways constructing and making HBV vector plasimids, which cannot express any structural genes ,by molecular biology technique. The new type HBV vector may be more security and applicable as a gene therapy vector.
Method
1 The original genes (Core, Polymerase, pre-S1, pre-S2, S and X) of HBV vector (pCH-S-GFP) were deleted by altering the start codon or adding stop codons, pCH-M5-GFP was successfuly constructed.
2 Deleting the GFP genes of pCH-S-GFP and pCH-M5-GFP, then inserting the hRLuc gene into the S region, pCH-S-hRLuc and pCH-M5-hRLuc were successfuly constructed.
3. To determine exogenous gene expression levels, after hepatocyte was cotransfected by the new plasimids and pCH3142, GFP and hRLuc were observed by fluorescence microscope or Luminometer.
4 To observed the ability of recombinant HBV replication, HBV DNA in cell lysate was extracted and southern blot was done to detect HBV replication intermediates, after plasmid cotransfection.
5 To detect the infectious capacity of the recombinant HBV on tree shrews hepatocyte tree shrews hepatocytes were infected by concentration and purification of reHBV particles from cell supernatant. The expression level of GFP would be observed after 1W.
Results
1 The three HBV vector plasmids were successfully constructed by gene recombination technology.
2 After the cells cotransfected by the mutation plasmid, expression level of GFP and hRLuc was stronger than the original plasmid.
3 Cotransfected by the mutation plasmid and pCH3142, the formation of HBV replication intermediates can be observed in Cell lysate by southern blot.
4 Expresstion of GFP can be observed after reHBV infected tree shrews hepatocytes.
Conclusion After the start codon and terminal codons of the HBV plasmids were mutated, HBV structure protein can not be expressed;Some report genes can be inserted into the S region of the new HBV vector plasmids, and stronger expression than the original plasmid;ReHBV can infect tree shrews hepatocytes.
Part two The first aspect application of the new type HBV vector---Stable cell line for secretion of replication-defective hepatitis B virus vector expressing blasticidin resistant gene
Objective:To construct a stable cell line with permanent secretion of recombinant hepatitis B virus(HBV)vector which express blasticidin resistant gene.
Method
1 Construction of HBV helper plasmid pcDNA3.1-CH3142 with G418-resistent non packing signal and HBV vector plasmids which express blasticidin resistant gene.
2 HepG2 cells were cotransfected with both the HBV vector expressing blasticidin resistant gene and the helper plasmids with G418-resistant gene. Cell clones were selected by the addition of both blasticidin and G418. Positive cell clones were gained.
3 Screening cell clones with high secretion of the HBV-Bsd recombinant hepatitis B virus particles by ELISA and dot blot hybridization
4 After culturing by the addition of both G418 and Bsd, regaining the higher level recombinant HBV cell line for secretion of replication-defective hepatitis B virus by Southernblot and Native Westernblot technology, and test the expression level of recombinant HBV through PCR
5 Application of PCR technology, wild-type hepatitis B virus and recombinant hepatitis B virus were identified and confirmed wild-type hepatitis B virus can not be formed in the new cell lines.
6 The complete recombinated HBV particles were tested by immune electron microscopy.
Result
1 Over 50 cell clones were formed through G418 screening after transfection.
2 36 cell clones were picked and expanded. HBV DNA in the supernatant was tested by dot blot hybridization with isotope labeled probe. Afterward the best 9 clones were selected
3 After maintaining for 15 generations (about 3 months), HBV DNA was proved by southern blot with isotope labeled probe. Confirmed the formation of RC DNA, According to the results, 3 cell lines of HBV-Bsd25,HBV-Bsd7, HBV-Bsd27 were chosen.
4 The quantity of HBV DNA of the three best cell lines (HBV-Bsd25, HBV-Bsd7, HBV-Bsd27) were 4.1*106, 3.6*106 and 1.2*106 copies/mL respectively.
5 No wild type HBV was detected.
6 Cesium chloride density gradient analysis in HBV-Bsd25 cell lines was done with the concentrated supernatant HBV virion, and enveloped recombinant HBV were proved by southern blot and HBsAg western blot.
7 Complete HBV particles were observed by electronic microscope.
Conclusion By the methods of HBV vector and HBV helper plasmids cotransfection, we could obtain the stable cell lines with permanent secretion of HBV virions and realized large preparation of recombinant HBV virions. This will contribute to the use of HBV vector for gene therapy. The recombinant HBV secretion stable cell line will also useful for the ongoing selection of HBV susceptible cell line.
Part three The second aspect application of the new type HBV vector plasmids---
An experimental study on rat liver cirrhosis model administrated with Adenovirus-HBV chimeric vector expressing collagenase II
No. 1 : construction and expression of adenovirus chimeric HBV vector in vitro
Objective To construct 3 adenovirus chimeric HBV vector plasmids which expressing different purpose gene, then observe the expressing levels of tMMP8 mRNA and RFP in vitro. To detect the expression of GFP in ECV304 and HepG2 with a CMV promoter and the HBV S promoter-driven GFP plasmids.
Method
1 Three new plasmids expressing purpose gene were successfully constructed.
2 To confirm the correctness of recombined adenovirus vector by PCR technology, the ideal gene fragments were obtained.
3 After three new correct plasmids were amplificated, extracted and purified, concentration determination and plaque-forming ability were detected.
4 After the three recombinant adenovirus infected HepaG2, RFP was detected by fluorescence microscopy, tMMP mRNA detected by RT-PCR technique.
2 After ECV304 and HepG2 were transfected with the plasmids of CMV promoter and the HBV S promoter-driven GFP, the expression of GFP was detected with fluorescence microscopy.
Result
1 HBV-adenovirus chimeric vector carrying different purpose genes were successfully constructed.
2 After recombinant adenovirus carrying MMP8 and tMMP8 infected hepatocytes, MMP8 and tMMP8 mRNA expression can be observed, recombinant adenovirus carrying RFP2 in hepatocytes can express RFP.
3 Defferent promoter-driven GFP plasmids transfected defferent cell lines, the level of GFP expression is defferent.
Conclusion Adenovirus vector can packaged HBV vector and expressing different purpose genes and formation HBV-adenovirus chimeric vector. After transfeced HepG2 cell lines, recombinated adenovirus vector can be successful expressing different purpose gene. HBV carrier s promoter-driven GFP in the vascular epithelial cells (ECV304) is low expression, in liver cell lines showed high expression. And CMV promoter-driven GFP in the vascular epithelial cells (ECV304) and liver cell lines showed high expression, suggesting that S promoter plasmid posseses hepatotropic.
No.2: An experimental study on rat liver cirrhosis model induced by TAA
Objective To explore the level of HGF/C-met, Liver histology, liver function and liver hydroxyproline on TAA induced rat liver cirrhosis model.
Method 42 male Wistar rats were fed with thioacetamide drinking water, preparation of liver fibrosis model. After the success of the model preparation, the rats were randomly divided into 7 groups of 6. Every two weeks killed a group of rats, collecting serum for liver function testing and detection of liver tissue hydroxyproline content, for HE and Sirius Red staining, and RT-PCR detected HGF and its receptor c-Met, and the other six normal rats were set as control group.
Result HGF/ c-Met mRNA expression was significantly higher than the normal control group. With the time to stop the extension of the application of TAA, liver function, histological changes, hydroxyproline content and HGF / c-Met mRNA expression level were returned to normal and a positive correlation was observed.
Conclusion:Rat liver cirrhosis models can be induced successfuly by TAA administrated for 20 weeks. After ceasing TAA, Liver histology, liver function and liver hydroxyproline have varying degrees of self-healing trend. Self-healing in the liver tissue at the same time, HGF and its receptor c-Met expression levels in varying degrees downward trend, however, no significant difference between groups. The occurrence of liver injury, an increase in HGF / c-Met expression level may be adopted to promote liver regeneration and reverse liver fibrosis
No.3: An experimental study on rat liver cirrhosis model administrated with Adenovirus-HBV chimeric vector expressing collagenase II
Objective Neutrophil collagenase (MMP-8) can degrade typeⅠcollagen specifically. Introduction of MMP-8 gene into liver cells could be an advantageous tool to degrade collagen. In this study, the aim is to explore the effectiveness of the typeⅠcollagen degradation, and the promotion of liver cell regeneration and improvement of liver function, to reduce liver fibrosis through treating cirrhosis rat model in vivo with Adenovirus-HBV chimeric vector expressing collagenase II
Method
1 Rat liver fibrosis model was prepared by 0.3% thioacetamide-induced drinking water.
2 Randomized and divided into groups and drug delivery 120 liver fibrosis model rats were randomly divided into four groups, were given Ad-CH-tMMP8, Ad-C-MMP8, Ad-CH-RFP2, tail vein injection of recombinant adenovirus, 1.5×1011 viral particles/Kg dose, one administration. Selecting another 30 rats as normal control group, administrated the normal water.
3 Ten rats in each group were sacrificed in 2W, 4W, 8W, respectively, and separate the serum, -20℃preservation. part of liver tissue were put in 10% of the formalin-fixed for histopathological examination, the remainder of the liver tissue were stored in liquid nitrogen.
4 Testing liver function by automatic biochemical analyzer, Hydroxyproline in liver tissue by acid hydrolysis method, the liver histopathological changes by HE, sirius red staining and immunohistochemical method, MMP8, HGF and c-Met and GAPDH mRNA by RT-PCR.
5 Statistical analysis. Statistical analysis was conducted by SAS statistical analysis software.
Result:
1 The expression level of MMP8 mRNA. MMP8 mRNA in the treatment group could be expressed in rat liver, but no MMP8 mRNA expression in the control group. The expression level of MMP8 mRNA was downward trend, with the extension of treatment time.
2 Liver function. As compared with two control groups, ALT in the two treatment groups were significantly decreased at 2 W, 4W and 8W(P <0.05), ALT in the two treatment groups was near to the normal group at 8W.
3 Indicators of liver fibrosis. HA and LN in the two treatment groups were significantly decreased at 2 W, 4W and 8W (P <0.05), HA and LN in the two treatment groups was near normal group at 8W.
4 Liver histology pathology Compared with the control group,pathology in the treatment group improve significantly,Inflammatory cell infiltration and the proliferation of tissue were significantly reduced. Hepatic lobules is damaged to a lesser extent. There is the phenomenon of liver regeneration
5 The quantity of Hydroxyproline. Hydroxyproline is an amino acid derived from collagen and elastin hydrolysis, accounting for about 14% of the weight of collagen. It is rare in other proteins. The quantity of hydroxyproline is varied with the severity of liver cirrhosis. At 2W application of the treatment of adenovirus, in the liver tissue, the hydroxyproline quantity was significantly lower than that of the control group
6 The expression level of HGF/cMet mRNA. HGF/cMet mRNA in the two treatment groups were significantly increased at 2 W, 4W and 8W(P <0.05) compared with control groups.
Conclusion:After application of recombinant adenovirus, liver tissue can express MMP8. The latter can effectively degrade collagen typeⅠ, reduce liver fibrosis, liver cells regenerate. The expression level of HGF / cMet mRNA was increased. This innovation is expected that MMP-8 gene therapy is brought into the clinical application of liver cirrhosis, it is a new strategy of treatment for reversing liver cirrhosis.
引文
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