摘要
第一部分
不同病理分期大鼠肝纤维化模型的建立
目的：
建立稳定的不同病理分期的大鼠肝纤维化动物模型,一方面为后期评估不同病变程度的干细胞治疗效果提供相对客观的受试对象,另一方面也可使目前国内外众多肝纤维化动物模型建立方法的标准化提供前期研究参考。
材料和方法：
以Sprague-Dawley (SD)大鼠为实验动物,以腹腔注射小剂量四氯化碳(CC14)+饮用乙醇溶液缓慢建立标准化大鼠肝纤维化动物模型。分别于建模后第2-15周时观察大鼠的血清透明质酸(Hyaluronic Acid, HA)、层粘连蛋白(Laminin, LN)、Ⅲ型前胶原氨端肽(Procollagen Ⅲ N-terminal Peptide, PⅢNP)、Ⅳ型胶原(Collagen Type Ⅳ，CⅣ)及结缔组织生长因子(Conective Tissue Growth Factor, CTGF)水平及肝脏病理活组织检查,按照肝纤维化病理分级标准对这些动物的肝纤维化程度进行评定。
结果：
通过小剂量、缓慢、个体化、联合给药法建模,可以观察到造模不同周次与其相对应的血清学指标和不同的病理学分期存在明确的对应规律。
结论：
该方法可标准化地建立S0-S4共5个肝纤维化病理分期的大鼠模型,且由于采用多种低毒性化学药物联合造模,在建模的较高成功率(89.33%)的同时保证了较低死亡率(17.3%)。
第二部分骨髓间充质干细胞体外扩增及鉴定
目的：
在体外成功地分离、培养和纯化Sprague—Dawley(SD)大鼠来源骨髓间充质干细胞(BMSCs)。
方法：
单纯贴壁法分离培养SD大鼠骨髓间充质干细胞,倒置显微镜观察细胞生长形态,流式细胞学检查骨髓间充质干细胞标志抗原的表达情况。
结果：
培养的BMSCs为多角形或梭形,人小形态相对均一,呈漩涡状排列生长。流式细胞学检查细胞表面标志抗原CD29、CD71、CD90高表达,而CD11b和CD45低表达情况。
结论：
采用单纯贴壁法可在体外成功地分离、培养和纯化大鼠BMSCs,为后续实验研究奠定了一部分实验基础。
第三部分CTGF基因RNA干扰慢病毒载体的构建与功能鉴定
目的：
通过相关检测确定可以采用RNA干扰对大鼠BMSCs进行基因修饰后,构建CTGF基因RNA干扰慢病毒载体感染大鼠BMSCs并获得较为稳定的细胞株,实验过程中逐步进行鉴定。
方法：
首先通过qPCR方法检测大鼠骨髓间充质干细胞(BMSCs)CTGF基因表达是否为高水平,以确定下调CTGF基因的操作方法是否可行。然后用“不同MOI值(Multiplicity of Infection)感染细胞测试(将病毒液按MOI=0,2,5,10,20,50,100,200,300加入培养的细胞,以确定合适的MOI值)”的方法确定慢病毒感染大鼠BMSCs细胞株最为适合的MOI值；构建针对靶基因的四个干扰质粒(pcDNATM6.2-X147-1、pcDNATM6.2-X147-2, pcDNATM6.2-X147-3、pcDN ATM6.2-X147-4),把这四个都有特定抗性表达序列的干扰质粒分别与高表达干扰序列--CTGF的载体(pCDNA3.1(+)-CTGF)共转染HEK293细胞,选择出干扰效果最佳的质粒,利用BP重组及LR重组的gateway技术,构建慢病毒表达载体；用构建的慢病毒表达载体和包装质粒(packaging mix)共转染293T细胞,包装病毒,收集病毒原液,超速离心浓缩,并测定滴度；将得到的同时有CTGF基因RNA干扰序列、GFP表达序列及氨苄青霉素抗性表达序列的慢病毒感染靶细胞,采用氨苄青霉素等抗性筛选法去除未经感染的阴性细胞。
结果：
靶基因CTGF有较高的表达；不同MOI值感染细胞测试证实MOI=200有较好的效果；干扰载体测序结果证实miRNA干扰载体的四个干扰质粒pcDNATM6.2-X147-1、pcDNATM6.2-X147-2、pcDNATM6.2-X147-3、 pcDNATM6.2-X147-4,高表达载体pCDNA3.1(+)-CTGF,'慢病毒载体pLenti6.3-X147-2,均构建成功；干扰载体在HEK293细胞中的干扰结果提示pcDNATM6.2-X147-2干扰效果最佳——干扰效率为81%。通过氨苄青霉素抗性筛选法去除了未经感染的细胞得到经CTGF基因RNA干扰慢病毒感染的大鼠BMSCs细胞株。
结论：
成功构建了大鼠BMSCs细胞株CTGF基因RNA干扰慢病毒载体Lenti6.3-X147-2,通过CTGF基因RNA干扰病毒载体感染大鼠BMSCs,得到了稳定的靶基因下调大鼠BMSCs细胞株。
第四部分CTGF基因RNA干扰慢病毒感染骨髓间充质干细胞对大鼠肝纤维化的治疗作用
目的：
观察CTGF基因RNA干扰慢病毒感染骨髓间充质干细胞对大鼠肝纤维化的治疗作用以及在不同肝纤维化程度下的治疗作用的差异。
方法：
选用健康雄性SD大鼠95只,按照论文第一部分“不同病理分期大鼠肝纤维化模型的构建”方法构建好动物模型,把得到的与时间依赖的规律性肝纤维化按照肝纤维化程度进行分级,在各级的大鼠中随机抽取实验对象组成：(1)空白对照组(A组)：接受开腹及肝尾叶切除、关腹手术,盲肠静脉注射1mlPBS溶液；(2)传统BMSCs治疗组(B组)：接受开腹及肝尾叶切除、关腹手术,盲肠静脉注射未经病毒感染的传统BMSCs细胞悬液1ml(1X107个细胞/m1)；(3)BMSCs-GFP示踪治疗组(空转组)(C组)：接受开腹及肝尾叶切除、关腹手术,盲肠静脉注射仅有GFP标记,没有RNA干扰序列的空白对照慢病毒感染的BMSCs-GFP细胞悬液1m1(1X107个细胞/m1)；(4)RNA干扰BMSCs治疗组(D组)：接受开腹及肝尾叶切除、关腹手术,盲肠静脉注射同时有GFP标记及CTGF-RNA干扰序列的空白对照慢病毒感染的BMSCs-RNAi-GFP细胞悬液1ml(1X107个细胞/ml)。手术及干细胞移植一周后处死动物,获取肝脏及血清标本,进行以下检测：(1)BMSCs移植样本肝脏组织荧光鉴定；
(2)酶联免疫吸附测定(Enzyme-linked Immuno Sorbent Assay,Elisa)法测定样本血清中肝纤维化四项指标：血清透明质酸(Hyaluronic Acid, HA)、层粘连蛋白(Laminin, LN)、Ⅲ型前胶原氨端肽(Procollagen Ⅲ N-terminal Peptide, PⅢNP)、Ⅳ型胶原(Collagen Type Ⅳ,CⅣ)及结缔组织生长因子(Conective Tissue Growth Factor, CTGF);
(3)样本肝脏组织病理切片HE染色；
(4)免疫组织化学(Immunohistochemistry，IHC)方法检测样本组织切片中CTGF蛋白表达的变化；
(5)实时荧光定量PCR法检测肝脏标本中CTGF基因mRNA表达水平；
(6) Western blot方法检测肝脏标本中CTGF蛋白的表达水平。
结果：
(1)转染了用GFP标记的BMSCs细胞后的荧光检测照片显示:BMSCs细胞经门静脉系注入肝纤维化模型后,能在大鼠肝脏中发现这些标记了GFP的BMSCs。
(2)血清中肝纤维化四项指标Elisa检测显示:BMSCs治疗的3组中这4个指标的表达量均显著下降。而RNAi干扰CTGF的BMSCs治疗的效果较BMSCs和GFP空转的BMSCs治疗效果要更好,血清四项的指标要更接近于正常值。
(3)病理切片HE染色结果显示：干扰CTGF(D组)后,]BMSCs治疗效果更加显著,小叶内变性细胞数、坏死灶面积,汇管间扩大程度及纤维索的数量均较模型组(A组)和传统BMSCs组(B组)和空转组(C组)有显著改善,空转组(C组)较模型组(A组)有很大改善,但是疗效没有CTGF干扰组(D组)显著。
(4)IHC检测CTGF蛋白表达显示：BMSCs治疗(B组、C组、D组)后表达CTGF的阳性细胞较模型组(A组)明显减少,其中传统BMSCs组(B组)和空转组(C组)改善的情况没有CTGF干扰组(D组)显著。D组中治疗前后的阳性表达区域(Average Optical Density, AOD)比值比较,治疗前CTGF表达阳性面积比为49.53±4.03,治疗后阳性面积为31.98±3.53,二者之间的P值为0.0083,达到了显著性水平。
(5) Realtime PCR检测显示：使用了不同BMSCs治疗的B组、C组和D组均可检测到CTGF基因mRNA表达水平的降低,其中采用了RNA干扰的D组效果最为显著。
(6) Western blot检测显示：随肝纤维化程度的增加,CTGF蛋白表达量也随之增加,而BMSCs治疗之后CTGF的表达量较之模型组有显著降低,RNAi干扰组的CTGF蛋白表达量为最低。
结论：
(1)肝脏组织荧光鉴定结果提示：经门静脉途径注射CTGF基因RNA干扰慢病毒感染BMSC后细胞可以定植在受损部位并可能生长并修复受损的肝脏组织。
(2)结果部分(2)至(6)的检测结果当中:①CTGF基因在mRNA及蛋白表达水平都随肝纤维化程度加重而上升的情况提示CTGF基因与肝纤维化病变进展密切相关系,推测CTGF基因可能参与了肝纤维化病变进展过程中某些关键的环节。②B组、C组、D组经BMSCs治疗后CTGF基因在转录及蛋白表达水平的明显下降提示BMSCs可能影响CTGF基因在转录、蛋白表达水平等某些关键的环节；其中RNA干扰BMSCs治疗后(D组)CTGF基因在转录及蛋白表达水平的下降及肝纤维化病变的减轻程度都较未经RNA干扰BMSCs治疗的B组、C组更为显著。
(3)在B组、C组、D组经BMSCs治疗后并未发现BMSCs治疗后加重肝纤维化的现象提示BMSCs加重肝纤维化可能只是特定条件下出现的偶然现象；但这并不能证明任何传统或经基因修饰的BMSCs移植对肝纤维化的治疗是一定有效或毫无风险的,采用CTGF基因RNA干扰BMSCs移植的治疗方法除了具有传统BMSCs的相关治疗作用外,还下调了BMSCs细胞内CTGF基因在转录及蛋白表达水平的表达,甚至改善了支配BMSCs分化方向的“小生境”从而可能在更大程度上在强化了BMSCs治疗肝纤维化作用的同时还限制了BMSCs在某些特定条件下可能出现的由于CTGF基因表达水平上调或使得肝纤维化加重的风险。
Part One
Establishment of a Standardized Liver Fibrosis Model with Different Pathological Stages in Rats
Objective:
To establish a standardized and stable liver fibrosis model with different pathological stages in rats. On one hand, it was necessary to lay foundation for later experimental procedures of this research, on the other hand, to explore a method to establish a standardized and stable liver fibrosis model creatively would contribute to evaluate other liver fibrosis model in rats.
Material and Methods:
The Sprague-Dawley (SD) rats were chose as experimental animals and endured intraperitoneal and subcutaneous injections of10%CC14liver oil solution at a dose of1□mL/kg administered twice a week for15weeks. The5%edible ethanol solution was used as the only drink for experimental rats. Five rats in the control group were sacrificed each week from the2nd to the1.5th week, and the blood-related indices(such as hyaluronic acid (HA), laminin (LN), procollagen Ⅲ N-terminal peptide (PIIINP), collagen type IV (CIV), conective tissue growth factor (CTGF) etc.) and pathological sections were detected for hepatic pathological grading analysis. The pathological grading was based on the criteria of Histological Grading and Staging of Chronic Hepatitis for Fibrosis.
Results:
Based on the mothods above, the regularity that the blood-related indices and the pathological grading changed along with the weeks after experiment can be observed.
Conclusion:
By taking the method above, the standardized and stable liver fibrosis model with different pathological stages (S0-S4) in rats could be established. Attributing to the chronic lesion induced by taking Low toxicity medicine long-termly and associatedly, the modeling success rate was89.33%, and the death rate was17.3%.
Part Two
Amplification And Identification in Vitro of Bone Marrow Mesenchymal Stem Cells
Objective:
To isolate, culture and purify establish the bone marrow mesenchymal stem cells(BMSCs) in vitro successfully.
Material and Methods:
The simple adherent method was adopted in isolating and culturing experiments of bone marrow mesenchymal stem cells(BMSCs). The inverted microscope was used to observe cells. The flow cytometry was used in checking cell mark antigens.
Results:
The morphological characteristics of the major of the BMSCs were unified into polygonal or fusiform, germinated into a spiral shape. Both high expression of CD29、CD71、CD90and low expression of CD11b and CD45were detected by the flow cytometry.
Conclusion:
The simple adherent method was a feasible way to isolate, culture and purify BMSCs successfully. It layed foundation for later experimental procedures of this research.
Part Three
Construction And Functional Identification of A Recombinant Lentiviral Vector Mediated RNAi Targeting CTGF Gene
Objective:
Afer confirming that it was feasible to modify the gene of rats by RNA interference, a recombinant lentiviral vector mediated RNAi targeting CTGF gene was constructed and was used to infect the BMSCs gotten from "Part Two". Then, the cell line infected and labled was cultured and become so stable that it could be indentified and named.
Material and Methods:
Firstly, whether it was high expression that the CTGF gene of the BMSCs gotten from " Part Two" should be tested by using qPCR method in order to confirm the later experimental plan. Secondly, the most calculated Multiplicity of infection (MOI) was confirmed after infected experiment by means of different MOI tested. Thirdly, everyone of the four interfering plasmids(pcDNATM6.2-X147-1, pcDNATM6.2-X147-2, pcDNATM6.2-X147-3, pcDNATM6.2-X147-4) and the vector with the hign expression of CTGF gene(pCDNA3.1(+)-CTGF) were chosen to infected the HEK293cell line. Fourthly, the most effective plasmid was used to construct the lentiviral vector by means of BPrecombination,LR recombination and gateway technique. Fifthly, the lentiviral vector and packaging plasmid (mix) were also used to infected293T cell line. NEXT, the lentiviral with viral titer determined was harvested after the viral fluid was concentrated by ultrafiltration. At last, to get the stable BMSCs cell line infected by the recombinant lentiviral which contains CTGF gene, GFP gene and Ampicillin resistance gene and to remove the other by means of resistance selection.
Results:
There were high expressions of CTGF gene in rats. The most calculated MOI value was200. The results of gene sequencing demonstrated that the four interferential plasmids(pcDNATM6.2-X147-1, pcDNATM6.2-X147-2, pcDNATM6.2-X147-3, pcDNATM6.2-X147-4), the vector with the high expression of CTGF gene(pCDNA3.1(+)-CTGF) and the lentiviral vector (pLenti6.3-X147-2) were constructed successfully. The effect of RNA interference confirmed that pcDNATM6.2-X147-2was the most effective plasmid and the interferential efficience was81%. The stable BMSCs cell line infected by the recombinant lentiviral which contains CTGF gene, GFP gene and Ampicillin resistance gene was harvested after resistance selection.
Conclusion:
The recombinant lentiviral vector mediated RNAi Targeting CTGF Gene (pLenti6.3-X147-2) were constructed successfully. A stable BMSCs cell line infected by the recombinant lentiviral was harvested.
Part Four
Therapeutic Effect of Bone Marrow Mesenchymal Stem Cells Infected by the RNAi Targeting CTGF Gene Mediated
by A Recombinant Lentivirus on Liver Fibrosis in Rats
Objective:
To observe the therapeutical impact of bone marrow mesenchymal stem cells infected by the RNAi targeting CTGF gene mediated by a recombinant lentivirus on liver fibrosis in rats and the difference between different pathological stages.
Material and Methods:
At first, A total of95male Sprague-Dawley (SD) were provided and established into a standardized and stable liver fibrosis model with different pathological stages in rats by means of "Part One". Next, the rats were divided into different parts with different pathological stages. The4groups were draw from different parts at random and designed as following:
Group A:received opening abdominal wall, hepatic caudate lobectomy, closing abdominal wall surgery and appendix intravenous injection of lml PBS solution.
Group B:received opening abdominal wall, hepatic caudate lobectomy, closing abdominal wall surgery and appendix intravenous injection of1ml BMSCs(without infected by virus) solution(1X107cells/ml).
Group C:received opening abdominal wall, hepatic caudate lobectomy, closing abdominal wall surgery and appendix intravenous injection of1ml BMSCs(infected the by lentivirus only with the high expression of EGFP gene sequence and without the RNAi targeting CTGF gene (pLenti6.3-X147-2) sequence) solution(1X107cells/ml).
Group D:received opening abdominal wall, hepatic caudate lobectomy, closing abdominal wall surgery and appendix intravenous injection of1ml BMSCs(infected the by lentivirus not only with the high expression of EGFP gene sequence and but also with the RNAi targeting CTGF gene (pLenti6.3-X147-2) sequence) solution(1X107cells/ml).
At the end, the animals were sacrificed at7day after operation, the liver and serum specimen labeled with numeral were harvested and analysed as following:
(1) To test with fluorescent identification of liver tissue harvested after BMSCs transplantation;
(2) To test the serum level of liver fibrosis markers (such as hyaluronic acid (HA), laminin (LN), procollagen Ⅲ N-terminal peptide (PⅢNP), collagen type Ⅳ (CⅣ), conective tissue growth factor (CTGF) etc.) ith enzyme-linked immuno sorbent assay (Elisa);
(3) To examine the pathologic change with pathological section of liver tissue and HE staining
(4) To examine the alteration of CTGF protein level in histotomy with Immunohistochemistry(IHC);
(5) To test the mRNA expression level of CTGF gene with Realtime PCR;
(6) To test the protein expression level of CTGF with Western blot.
Results:
(1) The photo of fluorescent identification of liver tissue harvested after BMSCs transplantation via the portal vein confirmed that the BMSCs could grow in diseased region and repair the damaged tissue.
(2) The serum level of liver fibrosis markers (such as hyaluronic acid (HA), laminin (LN), procollagen III N-terminal peptide (PⅢNP), collagen type Ⅳ (CIV), conective tissue growth factor (CTGF) etc.) of the3groups treated with BMSCs decreased obviously. And Group D held the biggest magnitude of the drop than Group B and C.
(3) The pathological section of liver tissue and HE staining confirmed that the degeneration cell number in hepatic lobule, the area of focal necrosis, expanding degree between portal regions and the quantity of fiber rope decreased obviously among the3groups:Group D> Group B≈Group C (the magnitude of the alteration).
(4) The results of Immunohistochemistry(IHC) demonstrated that the average optical density(AOD) value of the positive area of CTGF protein expression changed from49.53±4.03to31.98±3.53. The variation Reached the significant level(p=0.0083).
(5) The test of Realtime PCR confirmed that mRNA expression level of CTGF gene decreased distinctly in Group B and Group C, but no more obviously than Group D.
(6) Afer the protein expression level of CTGF was tested with Western blot, it was indicated that the CTGF protein increased with the severity of hepatic fibrosis, decreased obviously after BMSCs transplantation among the3groups:Group D> Group B≈Group C (the magnitude of the alteration).
Conclusion:
The BMSCs infected by the RNAi targeting CTGF gene mediated by a recombinant lentivirus could grow in diseased region and repair the damaged tissue. Moreover, the method of RNA interference down-regulated the over-expression of CTGF gene dramaticlly evoked by fibrosis likely. These were probable mechanism that the BMSCs infected by the RNAi targeting CTGF gene mediated by a recombinant lentivirus could alleviate liver fibrosis in rats in the test of fluorescent identification, the serum level of liver fibrosis markers (HA, LN, PⅢNP, CⅣ, CTGF, et al), pathologic change, IHC, Realtime PCR, Western blot, et al.
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