hNGFβ基因重组腺病毒表达载体的构建及其在星形胶质细胞和CCI动物中的作用研究
发布时间：2018-06-17 19:20
本文选题：同源重组 + 细菌 ； 参考：《华中科技大学》2005年博士论文
【摘要】： 第一部分细菌内同源重组构建含hNGFβ基因的腺病毒质粒 目的细菌内同源重组制备含hNGFβ基因的重组腺病毒质粒，在293细胞内包装后纯化重组腺病毒。方法将hNGFβ外源性核酸片段插入到经XmaⅠ与XbaⅠ酶切的pBluescriptⅡsk(+)，构建成转移质粒pBluescriptⅡsk(+)—hNGFβ，将其经Kpn I和Not I双酶切后，与经同样双酶切的穿梭质粒pShuttle-CMV定向重组，构建成穿梭质粒pShuttle-CMV-hNGFβ，将该穿梭质粒经PmeⅠ酶切后，，转化含腺病毒骨架质粒pAdEasy-1的感受态菌BJ5183，重组为pAdEasy-1-hNGFβ，酶切鉴定正确后，用Lipofect介导转染AD293细胞，包装为重组腺病毒Ad-hNGFβ后用PCR鉴定。透析纯化后的腺病毒采用紫外分光光度计进行滴度测定。结果线性化的pShuttle-CMV-hNGFβ转化含pAdEasy-1的感受态菌BJ5183，24h后获得了30％阳性重组质粒，经酶切得到一条大于20kb的大片段和一条4.5kb的特征性条带，PCR扩增出731bp片段。纯化后测得滴度为2.24×10~(12)OPU／L。结论应用细菌内同源重组能快速构建含hNGFβ基因的重组腺病毒载体pAdEasy-1-hNGFβ，酶切鉴定，包装为高滴度的重组腺病毒Ad—hNGFβ，为hNGFβ基因的应用研究奠定了基础。 第二部分Ad-hNGFβ在原代培养的脊髓星形胶质细胞的表达及其生物活性的研究 目的：以体外培养新生SD大鼠脊髓星形胶质细胞为靶细胞，证明其能表达有生物活性的目的基因产物，观察前期所重组的Ad-hNGFβ对靶细胞的存活及毒性作用，为下一步的在体研究打下基础。方法：从新生SD大鼠的脊髓组织中分离星形胶质细胞(Astrocyte，Ast)，应用DBEM／F-121∶1培养基进行原代培养及扩增，用胶质原纤维酸性蛋白(GFAP)免疫荧光法进行鉴定。用重组腺病毒Ad-hNGFβ(MOI为100)转染Ast后，用免疫组化法，初步测定培养3天时Ad-hNGβ的表达情况；用ELISA方法测定培养3d、6d及9d时，培养上清中NGFβ的含量；用噻唑蓝(MTT)比色法测定细胞OD_(570)值，分析细胞对Ad-hNGFβ的敏感性；用流式细胞仪对转染细胞进行倍体分析，观察对细胞的生长抑制作用。结果：从新生SD大鼠的脊髓组织中成功分离培养出星形胶质细胞，GFAP免疫荧光阳性。脊髓星形胶质细胞可被Ad-hNGFβ感染并表达hNGFβ；试验组hNGFβ表达量与对照组同期相比，均有显著差异，P＜0.01；并且随着细胞培养时间的延长，hNGFβ表达量下降，第9天与前两次比均有显著差异，P＜0.01。经噻唑蓝测定，试验组BTT的吸光值OD_(570)与对照组比有显著差异，P＜0.01。流氏细胞仪测得细胞凋亡与MTT结果同步。结论：Ad-hNGFβ能在原代培养的脊髓背角星形胶质细胞中表达，表达高峰时间约在第三天。MOI为100时无明显细胞毒作用，且能够增加细胞活性。 第三部分：鞘内注射Ad-hNGFβ对神经痛作用的研究 目的：研究鞘内注射Ad-hNGFβ对CCI大鼠疼痛的影响及机制。方法：建立CCI动物模型后，随机分为Ⅰ组：CCI术后立即L_5～L_6处鞘内注射Ad-hNGFβ；Ⅱ组：CCI术后立即L_5～L_6处鞘内注射人工脑脊液ACSF；Ⅲ组：假手术组，除不行坐骨神经结扎外，其它同Ⅱ组。测定术前、术后每4日各组大鼠足底热痛阈、机械痛阈值(最终结果以手术侧：非手术侧的比值表示)及行为学评分。分别于术后4天、7天、14天、28各天取8只动物麻醉后，4只灌注固定，取L_4～L_6段脊髓，免疫组化法测定脊髓背角痛物质SP、CGRP的变化；另4只不灌注固定，麻醉后直接断头处死，冰上快速取L_4～L_6脊髓匀浆后ELISA法测定hNGFβ的含量。结果：所有CCI动物术后0～28天均出现痛觉过敏，出现明显的疼痛行为学改变和热痛阈、机械痛阈的降低。CCI术后立即鞘内注射Ad-hNGF β动物自术后第4天开始热痛敏明显减轻，与同一时点CCI+ACSF组比，P＜0.05；整体疼痛行为学有所改善，但无显著差异；机械痛敏无明显改变。同时，注射Ad-hNGF β动物术后第4～28天脊髓hNGF β明显升高，与术前比，P＜0.01；术侧脊髓背角SP较ACSF组明显减少，P＜0.01或P＜0.05；CGRP无明显改变。结论：CCI术后立即L5-L6处鞘内注射Ad-hNGF β可明显减轻热痛敏，该改变与hNGF β在脊髓表达增加和SP表达减少有关。
[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k ( + ) - 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
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