發(fā)布時間：2018-06-17 19:20

  本文選題：同源重組 + 細(xì)菌　； 參考：《華中科技大學(xué)》2005年博士論文

【摘要】： 第一部分細(xì)菌內(nèi)同源重組構(gòu)建含hNGFβ基因的腺病毒質(zhì)粒 目的細(xì)菌內(nèi)同源重組制備含hNGFβ基因的重組腺病毒質(zhì)粒，在293細(xì)胞內(nèi)包裝后純化重組腺病毒。方法將hNGFβ外源性核酸片段插入到經(jīng)XmaⅠ與XbaⅠ酶切的pBluescriptⅡsk(+)，構(gòu)建成轉(zhuǎn)移質(zhì)粒pBluescriptⅡsk(+)—hNGFβ，將其經(jīng)Kpn I和Not I雙酶切后，與經(jīng)同樣雙酶切的穿梭質(zhì)粒pShuttle-CMV定向重組，構(gòu)建成穿梭質(zhì)粒pShuttle-CMV-hNGFβ，將該穿梭質(zhì)粒經(jīng)PmeⅠ酶切后，，轉(zhuǎn)化含腺病毒骨架質(zhì)粒pAdEasy-1的感受態(tài)菌BJ5183，重組為pAdEasy-1-hNGFβ，酶切鑒定正確后，用Lipofect介導(dǎo)轉(zhuǎn)染AD293細(xì)胞，包裝為重組腺病毒Ad-hNGFβ后用PCR鑒定。透析純化后的腺病毒采用紫外分光光度計進(jìn)行滴度測定。結(jié)果線性化的pShuttle-CMV-hNGFβ轉(zhuǎn)化含pAdEasy-1的感受態(tài)菌BJ5183，24h后獲得了30％陽性重組質(zhì)粒，經(jīng)酶切得到一條大于20kb的大片段和一條4.5kb的特征性條帶，PCR擴(kuò)增出731bp片段。純化后測得滴度為2.24×10~(12)OPU／L。結(jié)論應(yīng)用細(xì)菌內(nèi)同源重組能快速構(gòu)建含hNGFβ基因的重組腺病毒載體pAdEasy-1-hNGFβ，酶切鑒定，包裝為高滴度的重組腺病毒Ad—hNGFβ，為hNGFβ基因的應(yīng)用研究奠定了基礎(chǔ)。 第二部分Ad-hNGFβ在原代培養(yǎng)的脊髓星形膠質(zhì)細(xì)胞的表達(dá)及其生物活性的研究 目的：以體外培養(yǎng)新生SD大鼠脊髓星形膠質(zhì)細(xì)胞為靶細(xì)胞，證明其能表達(dá)有生物活性的目的基因產(chǎn)物，觀察前期所重組的Ad-hNGFβ對靶細(xì)胞的存活及毒性作用，為下一步的在體研究打下基礎(chǔ)。方法：從新生SD大鼠的脊髓組織中分離星形膠質(zhì)細(xì)胞(Astrocyte，Ast)，應(yīng)用DBEM／F-121∶1培養(yǎng)基進(jìn)行原代培養(yǎng)及擴(kuò)增，用膠質(zhì)原纖維酸性蛋白(GFAP)免疫熒光法進(jìn)行鑒定。用重組腺病毒Ad-hNGFβ(MOI為100)轉(zhuǎn)染Ast后，用免疫組化法，初步測定培養(yǎng)3天時Ad-hNGβ的表達(dá)情況；用ELISA方法測定培養(yǎng)3d、6d及9d時，培養(yǎng)上清中NGFβ的含量；用噻唑藍(lán)(MTT)比色法測定細(xì)胞OD_(570)值，分析細(xì)胞對Ad-hNGFβ的敏感性；用流式細(xì)胞儀對轉(zhuǎn)染細(xì)胞進(jìn)行倍體分析，觀察對細(xì)胞的生長抑制作用。結(jié)果：從新生SD大鼠的脊髓組織中成功分離培養(yǎng)出星形膠質(zhì)細(xì)胞，GFAP免疫熒光陽性。脊髓星形膠質(zhì)細(xì)胞可被Ad-hNGFβ感染并表達(dá)hNGFβ；試驗(yàn)組hNGFβ表達(dá)量與對照組同期相比，均有顯著差異，P＜0.01；并且隨著細(xì)胞培養(yǎng)時間的延長，hNGFβ表達(dá)量下降，第9天與前兩次比均有顯著差異，P＜0.01。經(jīng)噻唑藍(lán)測定，試驗(yàn)組BTT的吸光值OD_(570)與對照組比有顯著差異，P＜0.01。流氏細(xì)胞儀測得細(xì)胞凋亡與MTT結(jié)果同步。結(jié)論：Ad-hNGFβ能在原代培養(yǎng)的脊髓背角星形膠質(zhì)細(xì)胞中表達(dá)，表達(dá)高峰時間約在第三天。MOI為100時無明顯細(xì)胞毒作用，且能夠增加細(xì)胞活性。 第三部分：鞘內(nèi)注射Ad-hNGFβ對神經(jīng)痛作用的研究 目的：研究鞘內(nèi)注射Ad-hNGFβ對CCI大鼠疼痛的影響及機(jī)制。方法：建立CCI動物模型后，隨機(jī)分為Ⅰ組：CCI術(shù)后立即L_5～L_6處鞘內(nèi)注射Ad-hNGFβ；Ⅱ組：CCI術(shù)后立即L_5～L_6處鞘內(nèi)注射人工腦脊液ACSF；Ⅲ組：假手術(shù)組，除不行坐骨神經(jīng)結(jié)扎外，其它同Ⅱ組。測定術(shù)前、術(shù)后每4日各組大鼠足底熱痛閾、機(jī)械痛閾值(最終結(jié)果以手術(shù)側(cè)：非手術(shù)側(cè)的比值表示)及行為學(xué)評分。分別于術(shù)后4天、7天、14天、28各天取8只動物麻醉后，4只灌注固定，取L_4～L_6段脊髓，免疫組化法測定脊髓背角痛物質(zhì)SP、CGRP的變化；另4只不灌注固定，麻醉后直接斷頭處死，冰上快速取L_4～L_6脊髓勻漿后ELISA法測定hNGFβ的含量。結(jié)果：所有CCI動物術(shù)后0～28天均出現(xiàn)痛覺過敏，出現(xiàn)明顯的疼痛行為學(xué)改變和熱痛閾、機(jī)械痛閾的降低。CCI術(shù)后立即鞘內(nèi)注射Ad-hNGF β動物自術(shù)后第4天開始熱痛敏明顯減輕，與同一時點(diǎn)CCI+ACSF組比，P＜0.05；整體疼痛行為學(xué)有所改善，但無顯著差異；機(jī)械痛敏無明顯改變。同時，注射Ad-hNGF β動物術(shù)后第4～28天脊髓hNGF β明顯升高，與術(shù)前比，P＜0.01；術(shù)側(cè)脊髓背角SP較ACSF組明顯減少，P＜0.01或P＜0.05；CGRP無明顯改變。結(jié)論：CCI術(shù)后立即L5-L6處鞘內(nèi)注射Ad-hNGF β可明顯減輕熱痛敏，該改變與hNGF β在脊髓表達(dá)增加和SP表達(dá)減少有關(guān)。
[Abstract]:Construction of an adenovirus plasmid containing hNGF - 尾 gene by homologous recombination in the first part of bacteria









The recombinant adenovirus containing hNGF - 尾 gene was prepared by homologous recombination in bacteria . The recombinant adenovirus was purified by recombinant adenovirus . The recombinant adenovirus vector pBluescrip鈪�sk ( + ) - hNGF 尾 was transformed into pShuttle - CMV - hNGF . The recombinant adenovirus vector pAdEasy - 1 - hNGF - 尾 was transformed into pAdEasy - 1 . The recombinant adenovirus vector pAdEasy - 1 - hNGF 尾 was transformed into pAdEasy - 1 . The recombinant adenovirus vector pAdEasy - 1 - hNGF 尾 was digested by restriction enzyme digestion .









Expression of second part Ad - hNGF - 尾 in primary cultured spinal astrocyte and its biological activity









Objective : To investigate the expression of Ad - hNG尾 in neonatal SD rat spinal cord tissue and to investigate the survival and toxic effects of Ad - hNGF on target cells .
The content of NGF 尾 in culture supernatant was determined by ELISA .
The cell OD _ ( 570 ) value was determined by MTT assay , and the sensitivity of cells to Ad - hNGF 尾 was analyzed .
The growth inhibition of transfected cells was observed by flow cytometry . Results : Astrocytes were successfully isolated from the spinal cord tissue of neonatal SD rats . GFAP immunofluoresence was positive . The astrocytes of spinal cord could be infected with Ad - hNGF and express hNGF .
The expression of hNGF in the test group was significantly different from that in the control group ( P < 0.01 ) .
Conclusion : Ad - hNGF can be expressed in primary cultured spinal dorsal horn astrocytes , and the expression peak time is about the third day . Conclusion : Ad - hNGF can be expressed in primary cultured spinal dorsal horn astrocytes .









The third part : study on the effect of Ad - hNGF on neuralgia









Objective : To study the effect and mechanism of Ad - hNGF on CCI rats . Methods : After establishing the CCI animal model , we randomly divided into two groups : group 鈪

本文編號：2032172