人多巴胺D2受体基因内含子区51103T/C多态性对基因转录活性的影响

目的探讨人多巴胺D2受体（DRD2）基因第3内含子区51103 bp位点单核苷酸多态性（rs2075652）对DRD2基因转录活性的影响,进一步揭示DRD2基因第3内含子区的单核苷酸多态性影响创伤后应激障碍发病风险的分子机制。方法以人全血基因组DNA为模板,扩增DRD2基因第3内含子区序列（399 bp）,51103 bp处分别为T或C,与报告基因载体pmir Glo相连接,构建重组荧光素酶表达载体,经限制性内切酶酶切及测序得以鉴定,然后分别与pRL-CMV共转染人胚肾癌细胞株HEK293,检测细胞中荧光素酶的相对表达量。结果双酶切及测序结果证实成功构建重组质粒pmir Glo-T/pmir Glo-C,荧光素酶检测结果显示,当第3内含子区51103 bp位点为C时荧光素酶活性显著高于为T时（P〈0.01）。结论成功构建了pmir Glo-T/pmir Glo-C报告基因重组质粒,DRD2基因第3内含子区rs2075652位点由T突变为C后,可能增强基因转录活性,该结果为进一步研究DRD2基因内含子区的功能奠定了基础。     

嗅球成鞘细胞植入大鼠挫伤脊髓内的迁移分布特征

目的 观察嗅球成鞘细胞(olfactory ensheathing cells,OECs)在损伤脊髓内的迁移分布与脊髓功能恢复的关系.方法 用NYU-impactorⅡ装置以10 g·25 mm损伤大鼠T10脊髓,1周后将急性分离、纯化、鉴定的绿色荧光蛋白(GFP)-大鼠OECs植入脊髓损伤部位及其首尾两侧,OECs的移植量为90 000/μl.在移植后13周时间内,镜下定性观察OECs在冰冻切片上的迁移分布特征,然后对其分布面积和长度进行半定量观察.脊髓运动功能用BBB评分法测定.结果 移植后早期OECs主要聚集在移植部位,并逐渐向周围迁移扩散,但主要沿脊髓纵轴向首尾方向扩展,脊髓空腔内也可见移植的OECs.OECs的分布面积和长度分别由术后1周时的1.33 mm2和4.23 mm逐步扩大到13周时的3.30 mm2和7.68 mm.与此同时,大鼠损伤脊髓的运动功能也得到逐步恢复.结论 OECs植入挫伤脊髓后可以迁移游走,且与大鼠脊髓运动功能恢复有一定关系.

Abstract：

Objective To observe the migration and distribution of OECs in injured spinal cord and discuss their relation with the recovery of spinal cord function. Methods The rats were contused by a force of 10 g · 25 mm with NYU-impactor at T10 level. The OECs acutely isolated from green fluorescence protein (GFP) rats were purified, identified and then transplanted into the injured site and the rostral and caudal parts of the spinal cord one week after injury, with total volume of the transplanted OECs for 90 000/μl. Within 13 weeks after transplantation, the migration and distribution of OECs were qualitatively observed on the cryo-sections under fluorescence light microscope. The area and the length of OECs distribution were semi-quantitatively determined. The locomotor function of the spinal cord was appraised by BBB score. Results OECs were located collectively in the transplanted site at early stage after transplantation and then spread gradually mainly along the long axis of the cord. OECs could be found in the cavity of the contused spinal cord. The area and the length of OECs distribution were increased from 1.33 mm2 and 4.23 mm respectively at one week to 3.30 mm2 and 7.68 mm respectively at 13 weeks after transplantation. In the meantime, the locomotor function was gradually improved. Conclusion OECs can migrate within the contused spinal cord, as may contribute to the recovery of locomotor function.     

GFP标记成年大鼠表皮神经嵴干细胞的分离培养与形态学特征

目的建立绿色荧光蛋白(GFP)标记大鼠表皮神经嵴干细胞(EPI-NCSC)的分离培养方法,为EPI-NCSC在体移植研究脊髓损伤修复奠定基础。方法分离成年GFP大鼠胡须毛囊,取毛囊隆突部贴附于胶原包被的培养板上。在培养第4天有大量细胞游出,取出贴附的毛囊隆突部,细胞传代培养。细胞采用SOX10和Nestin免疫细胞化学染色鉴定,并计算细胞纯度。结果 EPI-NCSC在胶原基质上从隆突部游出,细胞为圆形或梭形,呈强绿色荧光。免疫细胞化学染色SOX10和Nestin双阳性,纯度在99%以上。结论本实验方法简便易行,能分离出高纯度的GFP-EPI-NCSC,可作为一种有效的工具细胞用于损伤脊髓修复研究。     

嗅鞘细胞移植到大鼠损伤脊髓前后的形态学研究

目的:观察体外培养和植入损伤脊髓的自发绿色荧光嗅鞘细胞(Green fluorescent protein-olfactory ensheathing cells,GFP-OECs)的形态结构特征,研究绿色荧光蛋白(Green fluorescent protein,GFP)及脊髓损伤的病理环境对其形态的影响.方法:用酶消化法分离培养来自2.5月龄的绿色荧光蛋白转基因大鼠嗅球最外层的嗅鞘细胞,对培养细胞定期进行光镜、电镜及激光共聚焦显微镜观察并摄片,观察其在体外培养的形态特征.培养第10d,用显微外科技术将嗅鞘细胞移植到脊髓挫伤后的T10节段,分别在移植术后7、15 d取材,观察细胞植入损伤脊髓后光电镜下形态.结果:GFP-OECs在荧光显微镜下呈绿色强荧光,培养第7 d,为双极样(梭形或纺锤形)、三极样和扁圆形3种典型的细胞形态,以双极梭形为主;培养第10d,细胞形态为梭形,植入损伤脊髓后,移植细胞胞体呈长梭形,一端突起细长,与另一嗅鞘细胞接触,呈条索状生长.电镜下嗅鞘细胞核不规则,胞质内有丰富的粗面内质网、游离核糖体及线粒体.结论:GFP不影响嗅鞘细胞(Olfactory ensheathing cells,OECs)形态.在脊髓损伤处,OECs胞体呈长梭形,突起细长,与培养10d移植时比较,形态无显著改变,早期脊髓损伤病理环境不影响GFP-OECs形态.     

嗅鞘细胞移植后脊髓挫伤大鼠几种神经营养因子的表达变化

目的探讨嗅鞘细胞移植脊髓挫伤大鼠后,在其神经损伤及修复过程中三种神经营养因子（脑源性神经营养因子、神经生长因子、睫状神经营养因子）的表达变化及意义。方法体外分离培养大鼠嗅球嗅鞘细胞。40只健康SD大鼠,雌雄不拘,在移植前一星期使用NYU-撞击机,利用10g-25mm的致伤力,制作T10脊髓挫伤模型。40只脊髓挫伤大鼠随机分为OECs移植组和对照组（注射无血清培养液组）,又根据取材时间不同分为术后1天、7天、14天、21天组。动物处死前进行BBB评分评估后肢运动功能的变化。取手术部位头端和尾端0.5cm脊髓,采用免疫组织细胞化学染色检测技术检测三种神经营养因子（BDNF、CNTF、NGF）在各组脊髓中的表达变化。结果行为学评分随着损伤时间的推移呈小幅度增加,但后肢无运动功能恢复;免疫组织化学检测显示,嗅鞘细胞移植能部分改善大鼠脊髓灰质神经细胞的凋亡程度,延缓白质神经纤维的减少,促进髓鞘的修复与再生。与对照组比较,嗅鞘细胞移植治疗后脊髓组织中三种神经营养因子表达明显增加（P〈0.05）。结论嗅鞘移植能够部分改善脊髓损伤后脊髓组织的病理形态,促进脊髓组织中多种神经营养因子的表达。     

嗅鞘细胞移植后大鼠损伤脊髓神经生长因子的表达

目的 研究嗅鞘细胞移植对大鼠损伤脊髓内的神经生长因子(NGF)表达的影响,以从NGF角度探讨嗅鞘细胞移植修复大鼠脊髓损伤的机制.方法 48只SD大鼠用NYU -Ⅱ撞击机(10g-25 mm)损伤T10脊髓制作脊髓损伤(SCI)模型,随机分为嗅鞘细胞组、DMEM组各24只;另设立正常对照组6只.将GFP-嗅鞘细胞细胞悬液移植入嗅鞘细胞组大鼠损伤处,DMEM组用单纯的DMEM/F12液代替,正常对照组不做任何处理.移植术后1d、7d、14 d、21 d,用BBB评分法测定脊髓运动功能,RT - PCR方法比较各时间点NGF表达差异.移植术后第21天应用免疫组化比较嗅鞘细胞组、DMEM组、正常对照组大鼠脊髓损伤区NGF的表达差异.结果 移植后1d、7d、14 d、21 d,嗅鞘细胞移植组运动功能评分均高于同期DMEM组.NGF表达量在术后第1天最高,第7天达峰值,之后缓慢降低.移植后第21天,嗅鞘细胞移植组脊髓NGF表达量高于同期DMEM组和正常对照组.结论 嗅鞘细胞移植可上调损伤脊髓NGF的表达,从而促进了损伤脊髓的修复.     

Survival and number of olfactory ensheathing cells transplanted in contused spinal cord of rats

Objective： To observe the survival and the number of olfactory ensheathing cells （OECs） transplanted in the contused spinal cord, so as to provide a basis for further studying the biological action of OECs.Methods： The rat spinal cords were contused with NYU-impactor Ⅱ at T10 level by dropping a 10 g rod from a height of 25 mm. At the 1st week after injury, OECs isolated freshly from green fluorecense protein （GFP） of the rats were transplanted into the spinal cord at injured site and other two sites 1 mm apart from the caudal and rostral ends with the OECs number of 30000/μl×3 =90000. The survival and the number of OECs were qualitatively and semi-quantitatively observed under the fluorescense microscope from 1 week to 13 weeks after transplantation. The motor function of the cord was evaluated with BBB score.Results： GFP-OECs could survive at least for 13 weeks within the contused spinal cord. Their arrangement was from tight to loose and their number was decreased from 1 week to 13 weeks after injury. The average number of GFP-OECs was 536 at the 1st week, which was less than 1% of the number as compared with original transplantation. After then, the number of GFP-OECs was continually decreased,but the most obvious decrease was found during 1 week to 2 weeks. The extent of decrease at other time points was relatively mild. In contrast to the cell number, motor function of the cord was gradually recovered after transplantation.Conclusions： The survival and the number of GFPOECs are different between the animals and are affected by the pathological reaction of the host cord. Also it is related to the motor function recovery of the contused cord.     

人多巴胺D2受体基因内含子区51103 T/C多态性对基因转录活性的影响

目的：探讨人多巴胺D2受体(DRD2)基因第3内含子区51103 bp位点单核苷酸多态性(rs2075652)对DRD2基因转录活性的影响,进一步揭示DRD2基因第3内含子区的单核苷酸多态性影响创伤后应激障碍发病风险的分子机制。方法以人全血基因组DNA为模板,扩增DRD2基因第3内含子区序列(399 bp),51103 bp处分别为T或C,与报告基因载体pmirGlo相连接,构建重组荧光素酶表达载体,经限制性内切酶酶切及测序得以鉴定,然后分别与pRL-CMV共转染人胚肾癌细胞株HEK293,检测细胞中荧光素酶的相对表达量。结果双酶切及测序结果证实成功构建重组质粒pmirGlo-T/pmirGlo-C,荧光素酶检测结果显示,当第3内含子区51103 bp位点为C时荧光素酶活性显著高于为T时(P<0．01)。结论成功构建了pmirGlo-T/pmirGlo-C报告基因重组质粒,DRD2基因第3内含子区rs2075652位点由T突变为C后,可能增强基因转录活性,该结果为进一步研究DRD2基因内含子区的功能奠定了基础。