本文選題：日本血吸蟲(chóng) + 童蟲(chóng)細(xì)胞　； 參考：《中南大學(xué)》2012年博士論文

【摘要】：第一章3種選擇培養(yǎng)基用于日本血吸蟲(chóng)細(xì)胞培養(yǎng)的研究 [目的]探索3種選擇培養(yǎng)基對(duì)日本血吸蟲(chóng)(Schistosoma japonicum,S.j)細(xì)胞作選擇培養(yǎng),觀察和鑒定各自對(duì)細(xì)胞培養(yǎng)的效果,探索通過(guò)選擇性培養(yǎng)基培養(yǎng)獲得單一種類(lèi)血吸蟲(chóng)細(xì)胞的可能性。[方法]分別從感染家兔獲取S.j-12d童蟲(chóng)和42d成蟲(chóng),按常規(guī)方法制備細(xì)胞。采用生殖類(lèi)細(xì)胞改良培養(yǎng)基對(duì)S.j-12d童蟲(chóng)和S.j-42d成蟲(chóng)細(xì)胞進(jìn)行選擇培養(yǎng)；同時(shí)采用上皮細(xì)胞培養(yǎng)基和1640-40綜合培養(yǎng)基對(duì)S.j-12d童蟲(chóng)細(xì)胞進(jìn)行培養(yǎng)。觀察日本血吸蟲(chóng)細(xì)胞經(jīng)這3種選擇培養(yǎng)基培養(yǎng)后-般生長(zhǎng)狀況和形態(tài)特點(diǎn)；通過(guò)染色體核型分析、AKP染色、超微結(jié)構(gòu)觀察對(duì)培養(yǎng)細(xì)胞進(jìn)行鑒定；利用BrdU摻入法或3H-TdR摻入法檢測(cè)部分培養(yǎng)細(xì)胞的增殖能力,以及運(yùn)用Dot-ELISA方法檢測(cè)培養(yǎng)細(xì)胞的抗原性。[結(jié)果]日本血吸蟲(chóng)細(xì)胞經(jīng)這3種選擇性培養(yǎng)基培養(yǎng)后,均有不同程度的增殖,細(xì)胞分裂相明顯,并且細(xì)胞活力狀態(tài)良好。經(jīng)生殖類(lèi)細(xì)胞改良培養(yǎng)基培養(yǎng)的童蟲(chóng)和成蟲(chóng)來(lái)源細(xì)胞在早期分裂現(xiàn)象明顯,呈葡萄串樣生長(zhǎng)繁殖,形成細(xì)胞團(tuán)；第3周可見(jiàn)大量形態(tài)結(jié)構(gòu)相近的細(xì)胞呈半貼壁生長(zhǎng)狀態(tài)。對(duì)培養(yǎng)細(xì)胞進(jìn)行鑒定發(fā)現(xiàn)細(xì)胞有DNA合成,染色體具有單倍體和雙倍體兩種核型；AKP染色為強(qiáng)陽(yáng)性,超微結(jié)構(gòu)鑒定可見(jiàn)胞質(zhì)中含有大量囊泡狀小體,此為生殖類(lèi)細(xì)胞的特征之一；培養(yǎng)4周細(xì)胞開(kāi)始出現(xiàn)退化死亡。日本血吸蟲(chóng)童蟲(chóng)細(xì)胞經(jīng)上皮細(xì)胞培養(yǎng)基培養(yǎng)后的鑒定結(jié)果顯示：培養(yǎng)3d的細(xì)胞出現(xiàn)類(lèi)上皮樣細(xì)胞生長(zhǎng),培養(yǎng)1周呈半貼壁生長(zhǎng)；細(xì)胞形態(tài)多呈長(zhǎng)梭形,結(jié)構(gòu)完整,核質(zhì)分明,細(xì)胞核清晰可見(jiàn),核仁明顯；超微結(jié)構(gòu)顯示細(xì)胞膜完整,細(xì)胞器無(wú)擴(kuò)張、水腫現(xiàn)象,核仁和核膜清晰；并且童蟲(chóng)細(xì)胞成分能被血吸蟲(chóng)細(xì)胞免疫血清所識(shí)別。根據(jù)細(xì)胞的形態(tài)特征和抗原性,初步認(rèn)為經(jīng)上皮細(xì)胞培養(yǎng)基培養(yǎng)的童蟲(chóng)細(xì)胞為上皮類(lèi)細(xì)胞。用1640-40綜合培養(yǎng)基培養(yǎng)的日本血吸蟲(chóng)細(xì)胞經(jīng)鑒定后結(jié)果顯示：培養(yǎng)細(xì)胞分裂相多樣,培養(yǎng)早期(30d)細(xì)胞狀態(tài)和活力較好,增殖相明顯；超微結(jié)構(gòu)觀察顯示培養(yǎng)30d細(xì)胞形態(tài)結(jié)構(gòu)基本正常,進(jìn)行細(xì)胞活力檢測(cè)達(dá)到70%；細(xì)胞增殖試驗(yàn)結(jié)果顯示細(xì)胞具有一定的增殖能力；染色體分析符合血吸蟲(chóng)二倍體核型；對(duì)細(xì)胞進(jìn)行凍存復(fù)蘇發(fā)現(xiàn)基本保持細(xì)胞原有的特性和活力。[結(jié)論]日本血吸蟲(chóng)混合細(xì)胞經(jīng)生殖類(lèi)細(xì)胞改良培養(yǎng)基和上皮細(xì)胞培養(yǎng)基培養(yǎng)后,能使混合細(xì)胞中類(lèi)似生殖類(lèi)細(xì)胞和上皮細(xì)胞進(jìn)行選擇性增殖,并具有該類(lèi)特定細(xì)胞的部分標(biāo)志性特征,可以達(dá)到初步分選細(xì)胞的目的,但連續(xù)培養(yǎng)時(shí)間不長(zhǎng)。1640-40綜合培養(yǎng)基能明顯促進(jìn)細(xì)胞增殖,改善細(xì)胞生長(zhǎng)狀態(tài),延長(zhǎng)細(xì)胞在體外的存活時(shí)間。 第二章SV40大T抗原基因重組腺病毒轉(zhuǎn)染日本血吸蟲(chóng)童蟲(chóng)與其細(xì)胞后的效果觀察 [目的]構(gòu)建含SV40大T抗原(SV40LT)基因的重組腺病毒表達(dá)載體,觀察重組腺病毒轉(zhuǎn)染日本血吸蟲(chóng)童蟲(chóng)及童蟲(chóng)細(xì)胞后SV40LT基因的表達(dá)情況,探討重組腺病毒載體轉(zhuǎn)染日本血吸蟲(chóng)童蟲(chóng)和童蟲(chóng)細(xì)胞的可能性,觀察轉(zhuǎn)染的外源SV40LT基因?qū)νx(chóng)和童蟲(chóng)細(xì)胞生長(zhǎng)狀況以及細(xì)胞增殖的影響。[方法]將國(guó)外贈(zèng)送的攜帶SV40LT基因的重組腺病毒質(zhì)粒(AdHu5-SV40LT)采用熱休克法重新轉(zhuǎn)化Stbl2感受態(tài)菌,獲得重組腺病毒質(zhì)粒。對(duì)質(zhì)粒用限制性?xún)?nèi)切酶酶切、PCR擴(kuò)增法進(jìn)行鑒定。鑒定正確后的質(zhì)粒經(jīng)Pac I酶切線性化后用脂質(zhì)體共轉(zhuǎn)染293A細(xì)胞,待出現(xiàn)細(xì)胞病變效應(yīng)(cell pathological effect, CPE)后收集細(xì)胞,經(jīng)反復(fù)凍融細(xì)胞離心收集細(xì)胞裂解上清,用氯化銫超速離心法對(duì)病毒上清進(jìn)行濃縮和純化,獲得具有感染性的重組腺病毒(Ad-SV40LT)。采用50%細(xì)胞培養(yǎng)感染法(50%cell culture infectious dose,CCID50)測(cè)定腺病毒的滴度。日本血吸蟲(chóng)尾蚴常規(guī)感染家兔獲得12d童蟲(chóng),一部分用改良841培養(yǎng)基培養(yǎng),另一部分按常規(guī)方法制備細(xì)胞,添加1640-40綜合培養(yǎng)基培養(yǎng)。兩者均培養(yǎng)3d后用重組腺病毒進(jìn)行轉(zhuǎn)染,繼續(xù)培養(yǎng)6-10d分別收集蟲(chóng)體和細(xì)胞,通過(guò)PCR、RT-PCR、 Western blotting和免疫組織化學(xué)方法檢測(cè)轉(zhuǎn)染童蟲(chóng)及童蟲(chóng)細(xì)胞中SV40LT基因的轉(zhuǎn)錄和表達(dá)情況。[結(jié)果]轉(zhuǎn)化Stbl2感受態(tài)菌后提取的AdHu5-SV40LT質(zhì)粒繹Pac Ⅰ酶切和PCR鑒定為日的質(zhì)粒。質(zhì)粒成功轉(zhuǎn)染293A細(xì)胞并可在293A細(xì)胞內(nèi)進(jìn)行有效的復(fù)制；從轉(zhuǎn)染293A細(xì)胞裂解上清中提取病毒DNA進(jìn)行PCR檢測(cè)證實(shí)含有SV40LT目的基因；同時(shí)Western blotting結(jié)果顯示在質(zhì)粒轉(zhuǎn)染的293A細(xì)胞中有SV40LT蛋白的表達(dá)；免疫組織化學(xué)染色也證實(shí)了同樣的結(jié)果。腺病毒轉(zhuǎn)染的293A細(xì)胞裂解上清經(jīng)氯化銫超速離心濃縮后測(cè)得病毒滴度為2.6×109cfu/ml。常規(guī)制備的S.j-12d童蟲(chóng)細(xì)胞用1640-40綜合培養(yǎng)基培養(yǎng)3d后可見(jiàn)細(xì)胞生長(zhǎng)狀態(tài)良好,部分細(xì)胞分裂相明顯。用重組腺病毒轉(zhuǎn)染的S.j-12d童蟲(chóng)及童蟲(chóng)細(xì)胞經(jīng)PCR檢測(cè)均擴(kuò)增出了外源SV40LT基因558bp的目的產(chǎn)物,進(jìn)一步用RT-PCR也證實(shí)了有目的基因mRNA的轉(zhuǎn)錄表達(dá)；Western blotting檢測(cè)結(jié)果顯示重組腺病毒轉(zhuǎn)染的童蟲(chóng)及其細(xì)胞內(nèi)有SV40LT蛋白的表達(dá)；免疫組織化學(xué)染色檢測(cè)也證實(shí)在蟲(chóng)體被膜下、口腹吸盤(pán)以及童蟲(chóng)細(xì)胞內(nèi)均有該蛋白的表達(dá)。病毒轉(zhuǎn)染后童蟲(chóng)活力未受影響,而童蟲(chóng)細(xì)胞轉(zhuǎn)染初期增殖較快,此后生長(zhǎng)逐漸減慢,細(xì)胞出現(xiàn)崩解、死亡。[結(jié)論]用質(zhì)粒AdHu5-SV40LT和脂質(zhì)體共轉(zhuǎn)染293A細(xì)胞后,成功獲得了具有感染能力的攜帶外源SV40LT基因的腺病毒；重組腺病毒轉(zhuǎn)染日本血吸蟲(chóng)童蟲(chóng)及童蟲(chóng)細(xì)胞后有目的基因SV40LT的轉(zhuǎn)錄和蛋白表達(dá),但轉(zhuǎn)染的外源SV40LT基因未能使童蟲(chóng)細(xì)胞獲得無(wú)限增殖能力而達(dá)到細(xì)胞永生化。本實(shí)驗(yàn)結(jié)果表明腺病毒能轉(zhuǎn)染日本血吸蟲(chóng)童蟲(chóng)及童蟲(chóng)細(xì)胞,并能將外源基因成功導(dǎo)入,為血吸蟲(chóng)的轉(zhuǎn)基因研究提供實(shí)驗(yàn)依據(jù)。
[Abstract]:Chapter 1 3 selective media for cell culture of Schistosoma japonicum
[Objective] to explore the selection culture of 3 kinds of selective medium for Schistosoma japonicum (S.j) cells, observe and identify the effect of each cell culture, explore the possibility of obtaining single type of Schistosoma cells through selective culture medium. [Methods] to obtain S.j-12d and 42d adult from infected rabbits, respectively, according to the routine. Methods the cells were prepared. The S.j-12d and S.j-42d adult cells were selected by reproductive cell improvement medium. At the same time, the epithelial cell culture medium and 1640-40 comprehensive medium were used to culture the S.j-12d worm cells. The growth status and morphological characteristics of Schistosoma japonicum cells were observed after the culture of these 3 kinds of culture medium. The culture cells were identified by chromosome karyotype analysis, AKP staining and ultrastructural observation. The proliferation ability of some cultured cells was detected by BrdU incorporation or 3H-TdR incorporation, and the antigenicity of the cultured cells was detected by Dot-ELISA method. [results] the cells of Schistosoma japonicum were cultured by these 3 selective medium. The cell division phase was obvious, and the cell viability was good. The early division phenomenon was evident in the early division of the children and the adult cells cultured on the improved cell culture medium. The cell clusters were produced in series, and the cells with similar morphologic structures were half adhered to the wall in the third week. The cells were identified as DNA synthesis, and the chromosomes were haploid and diploid two karyotypes; AKP staining was strong positive. Ultrastructural identification showed that the cytoplasm contained a large number of vesicular bodies, which was one of the characteristics of reproductive cells, and the cells began to degenerate for 4 weeks. The cells of Schistosoma japonicum by epithelia cells The results of the culture of culture medium showed that the cells in the culture of 3D appeared like epithelioid cells, and the cells were half attached to the wall for 1 weeks. The cell morphology was long spindle shaped, the structure was complete, the nucleolus was clear, the nucleus was clearly visible and the nucleolus were clear. The ultrastructure showed that the cell membrane was intact, the organelle was not dilated, the edema, nucleolus and nuclear membrane were clear. And the cell components of the children can be identified by the sera of Schistosoma cells. According to the morphological characteristics and antigenicity of the cells, the primary culture of the epithelioma cells cultured in the epithelial cell culture is epithelial cells. The results of the culture of Schistosoma japonicum cells cultured in 1640-40 comprehensive medium show that the cultures of the cultured cells are diverse and cultured. The state and vitality of 30d cells were better and the proliferation phase was obvious. Ultrastructural observation showed that the morphology and structure of the cultured 30d cells were basically normal, the detection of cell viability reached 70%, and the cell proliferation test showed that the cells had certain proliferation ability, and the chromosome analysis accords with the diploid karyotype of Schistosoma japonicum, and the cells were frozen and recovered. [Conclusion] the hybrid cells of Schistosoma japonicum can selectively proliferate like reproductive and epithelial cells in the mixed cells after the cultivation of the improved cell culture medium and epithelial cell culture medium, and have some characteristic characteristics of this kind of cell, which can be achieved. The aim of cell was preliminarily divided, but the continuous culture time of.1640-40 comprehensive culture medium could obviously promote cell proliferation, improve cell growth state and prolong the survival time of cells in vitro.
Second chapter SV40 large T antigen gene recombinant adenovirus transfection of Schistosoma japonicum larvae and its cells
[Objective] to construct a recombinant adenovirus expression vector containing SV40 large T antigen (SV40LT) gene and observe the expression of SV40LT gene in the transfected adenovirus of Schistosoma japonicum and the child's cell, and to explore the viability of the recombinant adenovirus vector transfection to the children and the cells of the Schistosoma japonicum. The growth status of the cell and the effect of cell proliferation. [Methods] the recombinant adenovirus plasmid (AdHu5-SV40LT), a recombinant adenovirus carrying the SV40LT gene, was converted into recombinant adenovirus vector by heat shock method, and the recombinant adenovirus plasmid was obtained by heat shock method. The plasmid was identified by restriction endonuclease digestion and PCR amplification. The correct plasmid was identified. After the tangent of Pac I enzyme, the 293A cells were co transfected with liposomes, and the cells were collected after the appearance of the cytopathic effect (cell pathological effect, CPE). The cells were collected by the centrifugation and centrifugation of the frozen thawing cells. The virus supernatant was concentrated and purified by cesium chloride overspeed centrifugation. The infected recombinant adenovirus (Ad-SV40LT) was obtained. 50%cell culture infectious dose (CCID50) was used to determine the titer of adenovirus. The cercariae of Schistosoma japonicum were routinely infected with 12D, some of which were cultured with modified 841 medium. The other part was prepared by conventional methods and cultured in 1640-40 comprehensive medium. Both of them were cultured for 3D and used recombinant adenovirus. Transfection, continue to culture 6-10d to collect insect body and cell respectively. Through PCR, RT-PCR, Western blotting and immunohistochemical method, the transcription and expression of SV40LT gene in the transfected children and children's cells were detected. [results] the AdHu5-SV40LT plasmids extracted from the Stbl2 receptive bacteria were transformed and the Pac I enzyme cut and PCR were identified as the plasmid of the day. The plasmid was successfully transfected into 293A cells and could be effectively replicated in the 293A cells. The PCR detection of the virus DNA from the transfected 293A cell cleavage supernatant confirmed the SV40LT target gene, and the Western blotting results showed the expression of SV40LT protein in the plasmid transfected 293A cells, and the immunohistochemical staining was also confirmed. The same results. The adenovirus transfected 293A cell cleavage supernatant was concentrated after cesium chloride overspeed centrifugation to detect the virus titer of 2.6 x 109cfu/ml., and the cell growth condition was good and the cell division phase was obvious. The S.j-12d worm transfected with recombinant adenovirus and the recombinant adenovirus transfected by recombinant adenovirus were in good condition. The target products of the exogenous SV40LT gene 558bp were amplified by PCR detection, and the transcriptional expression of the target gene mRNA was confirmed by RT-PCR, and the results of Western blotting detection showed that the recombinant adenovirus transfected with the expression of SV40LT protein in the cells and their cells, and the immunohistochemical staining was also proved to be in the insect body. Under the membrane, the expression of this protein was found in the stoma sucker and the cells of the worm. The vitality of the worm was not affected by the transfection of the virus, and the cell proliferation was faster in the early stage of transfection, then the growth slowed down gradually, and the cells disintegrated and died. [Conclusion] the infection ability was successfully obtained after transfecting 293A cells with plasmid AdHu5-SV40LT and liposomes. The adenovirus carrying the exogenous SV40LT gene; the recombinant adenovirus transfected with the target gene SV40LT of Schistosoma japonicum and the child's worm cells had the transcription and protein expression, but the transfected exogenous SV40LT gene failed to achieve the immortalization of the cells with unlimited proliferation ability. The results showed that the adenovirus could transfect the Schistosoma japonicum. The larvae and worm cells can successfully import exogenous genes, and provide experimental evidence for transgenic research of Schistosoma japonicum.

【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類(lèi)號(hào)】：R392.1

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