發(fā)布時間：2018-02-16 06:52

  本文關鍵詞： 異種移植 生殖細胞 轉基因 卵巢注射 精子載體 PAMAM-D α-1 2巖藻糖苷轉移酶 補體調節(jié)蛋白　出處：《天津醫(yī)科大學》2007年博士論文　論文類型：學位論文

【摘要】： 建立轉基因動物是異種移植研究中極為重要的工作基礎。我們采用以生殖細胞(卵細胞和精子細胞)為外源基因載體的新型轉基因技術建立轉基因動物，以簡化操作、降低成本、提高外源基因整合和表達的效率。 第一部分卵巢注射法建立hCD59、hCD55、HT單基因轉移小鼠的研究 目的探討卵巢注射法建立異種移植用轉基因動物的可行性。方法采用注射法將hC955、HT、hCD59基因重組質粒分別導入雌鼠卵巢內(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方法對目的基因整合進行初篩。對PCR出現(xiàn)特異條帶的小鼠基因組DNA進行Southern印跡雜交，進一步證實目的基因整合。抽取目的基因整合陽性小鼠的外周血，用逆轉錄-聚合酶鏈反應(RT-PCR)和流式細胞術(FCM)檢測目的基因表達。對FCM表達陽性的轉基因小鼠肝臟和腎臟進行免疫組織化學染色，觀察目的基因在器官中的表達分布情況。對表達目的基因的G_0代小鼠進行交配生產(chǎn)F1代小鼠，同法檢測F1代目的基因的整合與表達。分離3種轉基因小鼠的脾臟淋巴細胞，進行人血清溶破實驗。結果對15只雌性小鼠進行卵巢注射，共產(chǎn)生G_0代鼠130只。其中，注射HT基因重組質粒的小鼠產(chǎn)仔62只，注射hCD59基因重組質粒的小鼠產(chǎn)仔42只，注射hCD55基因重組質粒的小鼠產(chǎn)仔26只。經(jīng)PCR初篩后，Southern印跡雜交證實染色體中整合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檢測共20只G_0代小鼠陽性，包括3只hCD55小鼠、14只HT小鼠、3只hCD59小鼠。FCM檢測發(fā)現(xiàn)外周血單核細胞表達人H抗原、hCD55、hCD59的小鼠分別有9只、2只、2只，蛋白表達的效率為10.0％，高于受精卵顯微注射法的表達率。免疫組化檢測顯示，在轉基因小鼠的肝臟、腎臟等組織中均有相應外源基因的表達。G_0代小鼠交配后產(chǎn)下F1代小鼠37只，PCR檢測hCD55、HT、hCD59基因整合的小鼠分別為7只、6只、3只，F(xiàn)CM檢測表達的分別為0只、1只、1只。與對照組比較，三種轉基因小鼠的脾臟淋巴細胞耐受人血清溶破的能力均有所增強。結論采用卵巢注射法可以使針對異種移植的基因在小鼠體內(nèi)整合，并可實現(xiàn)RNA水平和蛋白質水平的表達，其整合與表達的效率要高于受精卵顯微注射法。三種轉基因構件均可遺傳給后代(F1代)，并在蛋白質水平表達。人血清溶破實驗證實，通過卵巢注射法制備的三種轉基因動物可以發(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移動物的可行性。方法采用hCD59／HT雙基因重組質粒和hCD59／hCD55／HT三基因重組質粒共同注射卵巢的方法，得到原代小鼠。PCR和Southern印跡雜交法檢測原代小鼠目的基因整合，F(xiàn)CM檢測目的基因表達。結果雙基因重組質粒共同注射4只雌鼠，獲得G_0代鼠31只。Southern印跡雜交證實僅出現(xiàn)hCD59／HT雙基因陽性鼠2只，無單基因整合鼠，僅1只鼠出現(xiàn)hCD59／HT共表達陽性，另1只鼠表達陰性。三基因重組質粒共同注射5只雌鼠，3胎共產(chǎn)生G_0代鼠137只，Southern印跡雜交證實僅出現(xiàn)hCD59／HT雙基因陽性鼠2只，無其它整合情況，表達均為陰性。結論多基因共注射卵巢法可以得到相應的轉基因動物，該方法確實可靠且周期較短。但多基因共注射后，基因之間會互相影響，整合與表達的效率比單基因注射者明顯降低，，提示其中有更復雜的機制參與，需要深入研究。 第三部分PAMAM-D對hCD55基因轉染豬精子細胞介導作用的研究 目的探討新型納米材料PAMAM-D對hCD55基因轉染豬精子細胞的介導作用。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復合物。取部分復合物酶切電泳。將復合物與處理后的1×10~6豬精子細胞共孵育2h，原位雜交法檢測hCD55對豬精子細胞的轉染效率，經(jīng)精子質量檢測工作站檢測孵育后的精子活力和精子畸形率。結果對PAMAM-D／DNA復合物進行酶切消化后，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，加入PAMAM-D后可以明顯提高各組豬精子細胞的轉染效率，且基本上隨著氮磷比的增加轉染效率也隨之增高。最高者(400ng，氮磷比20：1)可達對照組的167％(47.5％±3.5％vs 28.5％±1.5％)。經(jīng)精子質量檢測工作站檢測PAMAM-D對精子細胞活力和精子細胞畸形率沒有明顯影響(p＞0.05)。結論PAMAM-D作為載體可以提高目的基因對豬精子細胞的轉染效率，其對豬精子細胞無明顯毒性，增強了精子載體法的實用性，為生產(chǎn)異種移植用轉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.

【學位授予單位】：天津醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2007
【分類號】：R-332

【引證文獻】

相關博士學位論文 前1條

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

本文編號：1514917