發(fā)布時(shí)間：2018-02-16 06:52

  本文關(guān)鍵詞： 異種移植 生殖細(xì)胞 轉(zhuǎn)基因 卵巢注射 精子載體 PAMAM-D α-1 2巖藻糖苷轉(zhuǎn)移酶 補(bǔ)體調(diào)節(jié)蛋白　出處：《天津醫(yī)科大學(xué)》2007年博士論文　論文類型：學(xué)位論文

【摘要】： 建立轉(zhuǎn)基因動(dòng)物是異種移植研究中極為重要的工作基礎(chǔ)。我們采用以生殖細(xì)胞(卵細(xì)胞和精子細(xì)胞)為外源基因載體的新型轉(zhuǎn)基因技術(shù)建立轉(zhuǎn)基因動(dòng)物，以簡(jiǎn)化操作、降低成本、提高外源基因整合和表達(dá)的效率。 第一部分卵巢注射法建立hCD59、hCD55、HT單基因轉(zhuǎn)移小鼠的研究 目的探討卵巢注射法建立異種移植用轉(zhuǎn)基因動(dòng)物的可行性。方法采用注射法將hC955、HT、hCD59基因重組質(zhì)粒分別導(dǎo)入雌鼠卵巢內(nèi)，交配后產(chǎn)下原代(G_0代)小鼠。提取G_0代小鼠基因組DNA，采用PCR方法對(duì)目的基因整合進(jìn)行初篩。對(duì)PCR出現(xiàn)特異條帶的小鼠基因組DNA進(jìn)行Southern印跡雜交，進(jìn)一步證實(shí)目的基因整合。抽取目的基因整合陽(yáng)性小鼠的外周血，用逆轉(zhuǎn)錄-聚合酶鏈反應(yīng)(RT-PCR)和流式細(xì)胞術(shù)(FCM)檢測(cè)目的基因表達(dá)。對(duì)FCM表達(dá)陽(yáng)性的轉(zhuǎn)基因小鼠肝臟和腎臟進(jìn)行免疫組織化學(xué)染色，觀察目的基因在器官中的表達(dá)分布情況。對(duì)表達(dá)目的基因的G_0代小鼠進(jìn)行交配生產(chǎn)F1代小鼠，同法檢測(cè)F1代目的基因的整合與表達(dá)。分離3種轉(zhuǎn)基因小鼠的脾臟淋巴細(xì)胞，進(jìn)行人血清溶破實(shí)驗(yàn)。結(jié)果對(duì)15只雌性小鼠進(jìn)行卵巢注射，共產(chǎn)生G_0代鼠130只。其中，注射HT基因重組質(zhì)粒的小鼠產(chǎn)仔62只，注射hCD59基因重組質(zhì)粒的小鼠產(chǎn)仔42只，注射hCD55基因重組質(zhì)粒的小鼠產(chǎn)仔26只。經(jīng)PCR初篩后，Southern印跡雜交證實(shí)染色體中整合HT、hCD55、hCD59基因的小鼠分別有21只、4只、9只，總整合率為26.2％，高于受精卵顯微注射法的整合率。經(jīng)RT-PCR檢測(cè)共20只G_0代小鼠陽(yáng)性，包括3只hCD55小鼠、14只HT小鼠、3只hCD59小鼠。FCM檢測(cè)發(fā)現(xiàn)外周血單核細(xì)胞表達(dá)人H抗原、hCD55、hCD59的小鼠分別有9只、2只、2只，蛋白表達(dá)的效率為10.0％，高于受精卵顯微注射法的表達(dá)率。免疫組化檢測(cè)顯示，在轉(zhuǎn)基因小鼠的肝臟、腎臟等組織中均有相應(yīng)外源基因的表達(dá)。G_0代小鼠交配后產(chǎn)下F1代小鼠37只，PCR檢測(cè)hCD55、HT、hCD59基因整合的小鼠分別為7只、6只、3只，F(xiàn)CM檢測(cè)表達(dá)的分別為0只、1只、1只。與對(duì)照組比較，三種轉(zhuǎn)基因小鼠的脾臟淋巴細(xì)胞耐受人血清溶破的能力均有所增強(qiáng)。結(jié)論采用卵巢注射法可以使針對(duì)異種移植的基因在小鼠體內(nèi)整合，并可實(shí)現(xiàn)RNA水平和蛋白質(zhì)水平的表達(dá)，其整合與表達(dá)的效率要高于受精卵顯微注射法。三種轉(zhuǎn)基因構(gòu)件均可遺傳給后代(F1代)，并在蛋白質(zhì)水平表達(dá)。人血清溶破實(shí)驗(yàn)證實(shí)，通過(guò)卵巢注射法制備的三種轉(zhuǎn)基因動(dòng)物可以發(fā)揮抗異種排斥的作用。 第二部分卵巢內(nèi)共注射建立hCD59／HT雙基因和hCD55／hCD59／HT三基因轉(zhuǎn)移小鼠的初步研究 目的探討卵巢內(nèi)多基因共注射建立多基因轉(zhuǎn)移動(dòng)物的可行性。方法采用hCD59／HT雙基因重組質(zhì)粒和hCD59／hCD55／HT三基因重組質(zhì)粒共同注射卵巢的方法，得到原代小鼠。PCR和Southern印跡雜交法檢測(cè)原代小鼠目的基因整合，F(xiàn)CM檢測(cè)目的基因表達(dá)。結(jié)果雙基因重組質(zhì)粒共同注射4只雌鼠，獲得G_0代鼠31只。Southern印跡雜交證實(shí)僅出現(xiàn)hCD59／HT雙基因陽(yáng)性鼠2只，無(wú)單基因整合鼠，僅1只鼠出現(xiàn)hCD59／HT共表達(dá)陽(yáng)性，另1只鼠表達(dá)陰性。三基因重組質(zhì)粒共同注射5只雌鼠，3胎共產(chǎn)生G_0代鼠137只，Southern印跡雜交證實(shí)僅出現(xiàn)hCD59／HT雙基因陽(yáng)性鼠2只，無(wú)其它整合情況，表達(dá)均為陰性。結(jié)論多基因共注射卵巢法可以得到相應(yīng)的轉(zhuǎn)基因動(dòng)物，該方法確實(shí)可靠且周期較短。但多基因共注射后，基因之間會(huì)互相影響，整合與表達(dá)的效率比單基因注射者明顯降低，，提示其中有更復(fù)雜的機(jī)制參與，需要深入研究。 第三部分PAMAM-D對(duì)hCD55基因轉(zhuǎn)染豬精子細(xì)胞介導(dǎo)作用的研究 目的探討新型納米材料PAMAM-D對(duì)hCD55基因轉(zhuǎn)染豬精子細(xì)胞的介導(dǎo)作用。方法將新型納米材料PAMAM-D(G_5)和線狀hCD55 DNA按照不同氮磷比(電荷比)制備PAMAM-D／hCD55復(fù)合物。取部分復(fù)合物酶切電泳。將復(fù)合物與處理后的1×10~6豬精子細(xì)胞共孵育2h，原位雜交法檢測(cè)hCD55對(duì)豬精子細(xì)胞的轉(zhuǎn)染效率，經(jīng)精子質(zhì)量檢測(cè)工作站檢測(cè)孵育后的精子活力和精子畸形率。結(jié)果對(duì)PAMAM-D／DNA復(fù)合物進(jìn)行酶切消化后，其中的DNA分子不被限制性內(nèi)切酶降解。經(jīng)原位雜交法檢測(cè)，加入PAMAM-D后可以明顯提高各組豬精子細(xì)胞的轉(zhuǎn)染效率，且基本上隨著氮磷比的增加轉(zhuǎn)染效率也隨之增高。最高者(400ng，氮磷比20：1)可達(dá)對(duì)照組的167％(47.5％±3.5％vs 28.5％±1.5％)。經(jīng)精子質(zhì)量檢測(cè)工作站檢測(cè)PAMAM-D對(duì)精子細(xì)胞活力和精子細(xì)胞畸形率沒(méi)有明顯影響(p＞0.05)。結(jié)論P(yáng)AMAM-D作為載體可以提高目的基因?qū)ωi精子細(xì)胞的轉(zhuǎn)染效率，其對(duì)豬精子細(xì)胞無(wú)明顯毒性，增強(qiáng)了精子載體法的實(shí)用性，為生產(chǎn)異種移植用轉(zhuǎn)基因豬提供一種可供參考的方法。
[Abstract]:Transgenic animal is a very important basic work in xenotransplantation. We adopt germ cells (egg and sperm cells) for the establishment of transgenic animal model of transgenic technology of exogenous gene carrier, to simplify the operation, reduce the cost, improve the efficiency of exogenous gene integration and expression.
The first part of ovary injection to establish hCD59, hCD55, HT single gene transfer mice
Objective to investigate the feasibility of ovary injection method to establish transgenic animal xenograft. Methods using injection method, hC955, HT, hCD59 gene recombinant plasmids were introduced into ovaries, mating after the birth of primary (G_0) mice. G_0 mice genomic DNA extraction, using PCR method of gene integration were screened. The PCR specific band of mouse genomic DNA by Southern blot hybridization, further confirmed that the target gene integration. Peripheral blood samples were collected from the target gene integration positive mice, using reverse transcriptase polymerase chain reaction (RT-PCR) and flow cytometry (FCM) to detect the expression of target gene expression. The positive transgenic mice were immunized with liver and kidney histochemical staining of FCM, observe the expression of target gene in organ distribution. The expression of target gene in G_0 generation mice were mated to produce F1 generation mice, the same method for the detection of F1 gene generation Integration and expression. Separation of 3 transgenic mouse spleen lymphocytes, human serum 1yse. Experimental results are only 15 ovarian injection on female mice, the mice produced 130 G_0 generation. The injection of recombinant plasmid of HT gene of mice born 62, injection of recombinant plasmid of hCD59 gene of mice born 42, recombinant injection of hCD55 plasmid in mice. 26 mice were born after screening by PCR, Southern blot analysis confirmed that HT, hCD55 chromosome hCD59 gene integration, the mice were 21, 4, 9, the total integration rate was 26.2%, higher than the integration rate of microinjection. Detected by RT-PCR 20 G_0 mice were positive, including 3 hCD55 mice and 14 HT mice and 3 hCD59 mice.FCM detected expression of H antigen in peripheral blood mononuclear cells hCD55, hCD59 mice were 9, 2, 2, the expression rate was 10%, higher than the zygote microinjection The rate of expression. Immunohistochemistry showed that the transgenic mice liver, kidney and other tissues have the corresponding expression of exogenous gene in.G_0 generation mice F1 generation 37 mice, PCR detection of hCD55, HT, integration of hCD59 gene in mice were 7, 6, 3, FCM. Expression of respectively 0, 1, 1. Compared with the control group, the ability of the three transgenic mouse spleen lymphocyte tolerance of human serum 1yse is enhanced. Conclusion the method of ovary injection can make for xenotransplantation gene integration in mice, and can realize the RNA expression level and protein level the efficiency of integration and expression were higher than that of microinjection. Three transgenic components can be inherited to offspring (F1), and the expression in protein level. Human serum 1yse experiments confirmed that three transgenic animal by ovary injection preparation can play against The effect of xenograft rejection.
A preliminary study on the establishment of hCD59 / HT double gene and hCD55 / hCD59 / HT three gene transfer mice in second parts of the ovary
Objective to explore the feasibility of establishing multi gene transfer animal ovary gene co injection. Method hCD59 / HT double gene recombinant plasmid and hCD59 / hCD55 / HT three gene recombinant plasmid co injection of ovarian, primary mouse.PCR and Southern blot detection of primary mouse gene integration, to detect the expression of FCM gene results. Double gene recombinant plasmid co injection of 4 rats, 31 rats received G_0.Southern blot analysis confirmed that there were only hCD59 / HT double gene positive 2 rats, no single gene integration in 1 rats, only hCD59 / HT co expression, another 1 rats of three recombinant expression. Coinjeoted into 5 rats, 3 fetal G_0 produced a total of 137 rats, Southern blot analysis confirmed that there were only hCD59 / HT double gene positive 2 rats, no other integration, expression were negative. Conclusion multi gene co injection method of ovary In order to get the corresponding transgenic animals, the method is reliable and short. But after CO injection, the genes will interact with each other. The efficiency of integration and expression is significantly lower than that of single gene injectors, suggesting that there are more complex mechanisms involved, which need further research.
Study on the mediating effect of the third part of PAMAM-D on the transfection of hCD55 gene to porcine spermatozoa
Objective to explore the new nano PAMAM-D on hCD55 gene transfected pig sperm cells mediated by a novel method of nano materials PAMAM-D (G_5) and linear hCD55 DNA according to the different ratio of nitrogen and phosphorus (charge ratio) to prepare PAMAM-D / hCD55 composites. The compounds were digested by restriction enzymes. The compound and after treatment 1 x 10~6 pig sperm cells were incubated with 2H, transfection efficiency detected by hCD55 in situ hybridization in pig sperm cells, the sperm quality inspection workstation detection after incubation of sperm motility and sperm deformity rate. The results of PAMAM-D / DNA composites were digested, DNA molecules are not restriction enzyme degradation. Detected by in situ hybridization, after joining PAMAM-D groups can significantly improve the transfection efficiency of pig sperm cells, and basically with the ratio of nitrogen and phosphorus increased transfection efficiency was also increased. The highest (400ng, N / P ratio 20:1) than the control group 167% (47.5% + 28.5% + 1.5% 3.5%vs). The sperm quality detection workstation detection of PAMAM-D on sperm viability and sperm deformity rate of cells had no significant effect (P > 0.05). Conclusion PAMAM-D as carrier can improve the gene transfection efficiency of pig sperm cells, no obvious toxicity on pig sperm cells, enhance the practical sperm vector method, provides a reference method for production of xenotransplantation transgenic pigs.

【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2007
【分類號(hào)】：R-332

【引證文獻(xiàn)】

相關(guān)博士學(xué)位論文 前1條

1 郭戰(zhàn)軍;以納米材料作載體改進(jìn)精子介導(dǎo)法建立異種移植供體模型的研究[D];天津醫(yī)科大學(xué);2010年

本文編號(hào)：1514917