本文選題：間充質(zhì)干細胞 + 臍帶��； 參考：《中國協(xié)和醫(yī)科大學》2008年博士論文

【摘要】： 研究背景:目前糖尿病已成為僅次于心腦血管病癥和癌癥的第三大死亡疾病,它是一種影響體內(nèi)胰島素和糖含量的疾病,共有兩種主要類型,分別為Ⅰ型和Ⅱ型,其共同特征是胰島β細胞損傷或不能產(chǎn)生足夠的胰島素或機體不能有效的利用胰島素,所以胰島素就被作為治療糖尿病的特效藥應(yīng)用于臨床。由于隨時需要注射胰島素,給患者帶來了極大的不便,目前國際上對糖尿病患者進行胰島移植和基因治療研究成為了熱點。但面臨的主要問題是供體不足,免疫排斥,胰島素基因的轉(zhuǎn)染效率和表達調(diào)控等。人臍帶來源的間充質(zhì)干細胞可以較易獲得和純化,并且易于基因修飾,可以將外源性基因轉(zhuǎn)染間充質(zhì)干細胞,建立一種安全的基因治療的“細胞載體”。研究表明間充質(zhì)干細胞還有免疫抑制功能,它不但在體外可以抑制T淋巴細胞增殖反應(yīng),在體內(nèi)實驗中也表現(xiàn)出了類似免疫抑制劑的作用,所以利用它作為細胞載體可以避免免疫排斥問題。并且我們利用基因重組的方法將胰島素原基因進行改造,使其可以在非β細胞(間充質(zhì)干細胞)中高效表達。相信我們的研究可以為Ⅰ型糖尿病的治療提供一些有力的實驗依據(jù)。目的:將人胰島素原基因進行兩個部位的突變,通過腺病毒介導將其轉(zhuǎn)染到人臍帶間充質(zhì)干細胞中,研究其表達分泌人胰島素的能力。方法:應(yīng)用RT-PCR的方法從健康流產(chǎn)胎兒胰腺組織中擴增pINS的cDNA序列,利用重疊延伸PCR方法對pINS基因在A鏈-C肽和C肽-B鏈的連接處進行兩個部位的突變,產(chǎn)生Furin蛋白酶酶切位點。采用AdEasy~(TM)系統(tǒng)構(gòu)建重組腺病毒pAdINS、pAdINS-M2和pAdGFP。分離培養(yǎng)人臍帶間充質(zhì)干細胞,并對其生長特性,免疫表型,分化能力及Furin蛋白酶的表達進行鑒定。pAdINS、pAdINS-M2和pAdGFP感染人臍帶間充質(zhì)干細胞后,利用熒光顯微鏡和流式細胞儀檢測它們的感染效率,并確定最佳的感染復數(shù)(multiplicities of infection,MOI)。以感染復數(shù)為100的pAdINS、pAdINS-M2或pAdGFP感染人臍帶間充質(zhì)干細胞1、3、5、7、10天后,RT-PCR檢測人胰島素原基因在人臍帶間充質(zhì)干細胞中表達;Western blot檢測成熟人胰島素和人C-肽在人臍帶間充質(zhì)干細胞中的表達;免疫熒光實驗檢測感染后48小時人胰島素在人臍帶間充質(zhì)干細胞中的表達:ELISA實驗檢測感染后1、3、5、7、10天人胰島素和人C-肽的分泌。結(jié)果:自健康流產(chǎn)胎兒胰腺組織中擴增出364 bp的pINS cDNA,連接入穿梭質(zhì)粒中,構(gòu)建重組腺病毒。pAdINS-M2經(jīng)擴增純化后滴度達到2.57×10~(10) PFU/mL,pAdINS的滴度為1.25×10~(10)PFU/mL,對照病毒AdGFP為4.85×10~9 PFU/mL。成功的從臍帶組織中分離培養(yǎng)出間充質(zhì)干細胞,繪制生長曲線,約3天擴增一代。細胞周期檢測,大部分細胞都處于G0-G1期(占94.39%),小部分處于G2-M期(4.15%)和S期(1.45%)。在合適的誘導培養(yǎng)條件下,分離培養(yǎng)的人臍帶間充質(zhì)干細胞能夠分化為脂肪細胞和成骨細胞。應(yīng)用RT-PCR方法在人臍帶間充質(zhì)干細胞中檢測到了Furin蛋白酶的存在,表明我們構(gòu)建的含F(xiàn)urin蛋白酶酶切位點的人胰島素原基因在此細胞中可以被切割產(chǎn)生成熟的人胰島素。通過重組腺病毒pAdINS、pAdINS-M2和pAdGFP以不同感染復數(shù)感染人臍帶間充質(zhì)干細胞,利用熒光顯微鏡和流式細胞儀檢測感染效率,確定MOI 100為最佳的感染復數(shù)。以MOI=100的pAdINS、pAdINS-M2或pAdGFP感染人臍帶間充質(zhì)干細胞1、3、5、7、10天后,RT-PCR方法檢測到在pAdINS和pAdINS-M2感染的UC-MSCs中人胰島素原基因都有表達;Western blot的結(jié)果顯示pAdINS感染的UC-MSCs表達人胰島素原,而pAdINS-M2感染的UC-MSCs中檢測到了人胰島素和C.肽,pAdGFP感染的UC-MSCs中兩者都沒有檢測到表達。免疫熒光也檢測了pAdINS-M2感染后48小時人胰島素和C-肽在人臍帶間充質(zhì)干細胞胞漿中表達:ELISA檢測感染后1、3、5、7、10天培養(yǎng)上清中人胰島素和C-肽的濃度。結(jié)果顯示,自感染后24小時目的基因就開始表達,第3-5天表達量達高峰,一周后表達有所下降。與RT-PCR和Western blot結(jié)果一致。結(jié)論:成功構(gòu)建含有Furin蛋白酶切位點的人胰島素原基因腺病毒重組體(pAdINS-M2);分離培養(yǎng)了人臍帶源間充質(zhì)干細胞,并對其進行了鑒定;重組腺病毒pAdINS-M2感染人臍帶間充質(zhì)干細胞后,表達分泌成熟人胰島素和C-肽。可以為Ⅰ型糖尿病的治療提供一些有力的實驗依據(jù)。
[Abstract]:Background: diabetes has become the third most fatal disease after cardio cerebrovascular disease and cancer. It is a disease that affects insulin and sugar content in the body. There are two main types, type I and type II, which are characterized by islet beta cell damage or failure to produce sufficient insulin or the body is not effective. Insulin is used as a special drug for the treatment of diabetes. Because of the need for insulin injection at any time, it has brought great inconvenience to the patients. Now, the international study of islet transplantation and gene therapy for diabetic patients has become a hot spot. But the main problem is the deficiency of donor, immune rejection, and pancreas. The transfection efficiency and expression regulation of islin gene. Mesenchymal stem cells from human umbilical cord derived mesenchymal stem cells can be easily obtained and purified, and are easily genetically modified. Exogenous genes can be transfected into mesenchymal stem cells to establish a "cell carrier" for a safe gene therapy. It is also shown that mesenchymal stem cells have immunosuppressive function, and it also shows that mesenchymal stem cells also have immunosuppressive function. It can not only inhibit the proliferation of T lymphocyte in vitro, but also show the effect of immunosuppressant in the experiment in vivo, so using it as a cell carrier can avoid the problem of immune rejection. And we use gene recombination method to modify the proinsulin gene, so that it can be used in non beta cells (mesenchymal stem cells). We believe our study can provide some powerful experimental basis for the treatment of type I diabetes. Objective: to transfect the human proinsulin gene in two sites and transfect it into human umbilical cord mesenchymal stem cells via adenovirus to study the ability to express human insulin. Methods: the application of RT-PCR. The cDNA sequence of pINS was amplified from the fetal pancreas of healthy abortion, and the pINS gene was mutated at the junction of A chain -C peptide and C peptide -B chain by overlapping extension PCR method, and the Furin protease tangent site was produced. The recombinant adenovirus pAdINS was constructed by AdEasy~ (TM) system, and the human umbilical cord mesenchyme was isolated and cultured. Mesenchymal stem cells, and identification of their growth characteristics, immunophenotype, differentiation ability and expression of Furin protease,.PAdINS, pAdINS-M2 and pAdGFP infected human umbilical cord mesenchymal stem cells, using fluorescence microscopy and flow cytometry to detect their infection efficiency, and determine the optimal number of multiplicities of infection (MOI). PAdINS, pAdINS-M2 or pAdGFP infected human umbilical cord mesenchymal stem cells for 1,3,5,7,10 days, RT-PCR detected human proinsulin gene expression in human umbilical cord mesenchymal stem cells; Western blot was used to detect the expression of mature human insulin and human C- peptide in human umbilical cord mesenchymal stem cells; immunofluorescence test detected the infection for 48 hours after infection. The expression of human insulin in human umbilical cord mesenchymal stem cells: ELISA test was used to detect the secretion of insulin and human C- peptide in 1,3,5,7,10 days after infection. Results: 364 BP pINS cDNA was amplified from the fetal pancreas tissue from healthy abortion and joined into the shuttle plasmid, and the recombinant adenovirus.PAdINS-M2 was constructed and purified to reach 2.57 x 10~ (10) P after amplification and purification. The titer of FU/mL, pAdINS was 1.25 x 10~ (10) PFU/mL. Compared with the virus AdGFP of 4.85 * 10~9 PFU/mL., mesenchymal stem cells were isolated and cultured from the umbilical cord tissue successfully. The growth curve was plotted for about 3 days. Cell cycle detection, most of the cells were in G0-G1 period (94.39%), and the small part was in G2-M phase (4.15%) and S period (1.45%). The cultured human umbilical cord mesenchymal stem cells can differentiate into adipocytes and osteoblasts under the induced culture conditions. The presence of Furin protease in human umbilical cord mesenchymal stem cells is detected by RT-PCR method, indicating that the human proinsulin gene, which is constructed with the Furin protease cutting site, can be cut in this cell. Human umbilical cord mesenchymal stem cells were infected by recombinant adenovirus pAdINS, pAdINS-M2 and pAdGFP with different infection complex numbers. The infection efficiency was detected by fluorescence microscopy and flow cytometry, and MOI 100 was the best complex number of infection. Human umbilical cord mesenchymal stem cells were infected with MOI=100 pAdINS, pAdINS-M2 or pAdGFP. 1,3,5,7,10 days later, the RT-PCR method detected the expression of human proinsulin gene in both pAdINS and pAdINS-M2 infected UC-MSCs; Western blot results showed that UC-MSCs expressed human proinsulin in pAdINS infection, while both human insulin and C. peptides were detected in UC-MSCs pAdINS-M2 infection, both of which were not detected. Immunofluorescence also detected the expression of insulin and C- peptide in human umbilical cord mesenchymal stem cell cytoplasm 48 hours after pAdINS-M2 infection: ELISA was used to detect the concentration of human insulin and C- peptide in the culture supernatant of 1,3,5,7,10 days after infection. The results showed that the target gene was expressed at the beginning of the infection 24 hours after infection, the expression level reached the peak at day 3-5, and a week later The results were in accordance with the results of RT-PCR and Western blot. Conclusion: the recombinant human proinsulin gene adenovirus recombinant (pAdINS-M2) containing Furin protease site was successfully constructed, and the human umbilical cord derived mesenchymal stem cells were isolated and cultured, and the recombinant adenovirus pAdINS-M2 infected human umbilical cord mesenchymal stem cells and expressed and secreted. Mature human insulin and C- peptide can provide some powerful experimental evidence for the treatment of type I diabetes.
【學位授予單位】：中國協(xié)和醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2008
【分類號】：R587.1;R346

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