中华神经科杂志
中華神經科雜誌
중화신경과잡지
Chinese Journal of Neurology
2015年
8期
697-703
,共7页
刘凯%阴晓峰%相恒伟%邓文帅%孙鹏
劉凱%陰曉峰%相恆偉%鄧文帥%孫鵬
류개%음효봉%상항위%산문수%손붕
帕金森病%间质干细胞%神经组织蛋白质类%慢病毒属%转染%疾病模型,动物
帕金森病%間質榦細胞%神經組織蛋白質類%慢病毒屬%轉染%疾病模型,動物
파금삼병%간질간세포%신경조직단백질류%만병독속%전염%질병모형,동물
Parkinson's disease%Mesenchymal stem cells%Nerve tissue proteins%Lentivirus%Transfection%Disease models,animal
目的 探讨慢病毒介导的artemin(ARTN)基因修饰的骨髓间充质干细胞(MSCs)对帕金森病模型大鼠潜在性治疗作用及对脑内相关蛋白表达的影响.方法 分离培养大鼠MSCs,将携带ARTN的慢病毒载体转染MSCs;6-羟多巴胺(6-OHDA)制备帕金森病大鼠模型,按随机数字表法随机分为假手术(Sham)组、帕金森病组、移植MSCs(MSCs)组、移植空病毒转染的MSCs(LV-MSCs)组、移植慢病毒介导的ARTN基因修饰的MSCs (LV-ARTN-MSCs)组,对帕金森病大鼠左侧纹状体进行细胞移植;术后2、4、6、8周,腹腔注射阿扑吗啡,进行行为学检测;采用Western印迹及免疫荧光法检测各组大鼠黑质酪氨酸羟化酶(TH)表达变化,移植ARTN修饰的MSCs在大鼠脑组织内表达;高效液相色谱仪检测各组大鼠纹状体内多巴胺、二羟基苯乙酸、高香草酸含量.结果 LV-ARTN-MSCs组在阿扑吗啡诱发下30 min内旋转圈数(179.33±18.74)明显低于帕金森病组、MSCs组和LV-MSCs组(235.83±18.95、203.67±11.50和206.33±11.86;q=8.828,P＜0.01;q=3.802,P＜0.05;q=4.219,P＜0.05).LV-ARTN-MSCs组大鼠黑质TH阳性细胞百分比(64.05％±5.49％)显著高于帕金森病组(34.18％±3.35％)、MSCs组(52.59％±4.48％)和LV-MSCs组(50.57％±4.41％;q=13.280、5.135、6.028,均P＜0.01),黑质TH蛋白表达也明显提高,同时慢病毒介导ARTN修饰的MSCs可以在帕金森病大鼠脑内存活至少6周,主要集中在移植侧的纹状体区.细胞移植8周后,MSCs组(2.34±0.54)、LV-MSCs组(2.28±0.45)、LV-ARTN-MSCs组(6.54±0.51)与帕金森病组(0.87±0.07)大鼠相比,纹状体多巴胺表达明显提高(q =5.233,P＜0.05;q=5.020,P＜0.01;q=20.190,P＜0.01),以LV-ARTN-MSCs组最明显.结论 纹状体移植慢病毒介导ARTN基因修饰的MSCs对帕金森病模型大鼠有一定的治疗作用.
目的 探討慢病毒介導的artemin(ARTN)基因脩飾的骨髓間充質榦細胞(MSCs)對帕金森病模型大鼠潛在性治療作用及對腦內相關蛋白錶達的影響.方法 分離培養大鼠MSCs,將攜帶ARTN的慢病毒載體轉染MSCs;6-羥多巴胺(6-OHDA)製備帕金森病大鼠模型,按隨機數字錶法隨機分為假手術(Sham)組、帕金森病組、移植MSCs(MSCs)組、移植空病毒轉染的MSCs(LV-MSCs)組、移植慢病毒介導的ARTN基因脩飾的MSCs (LV-ARTN-MSCs)組,對帕金森病大鼠左側紋狀體進行細胞移植;術後2、4、6、8週,腹腔註射阿撲嗎啡,進行行為學檢測;採用Western印跡及免疫熒光法檢測各組大鼠黑質酪氨痠羥化酶(TH)錶達變化,移植ARTN脩飾的MSCs在大鼠腦組織內錶達;高效液相色譜儀檢測各組大鼠紋狀體內多巴胺、二羥基苯乙痠、高香草痠含量.結果 LV-ARTN-MSCs組在阿撲嗎啡誘髮下30 min內鏇轉圈數(179.33±18.74)明顯低于帕金森病組、MSCs組和LV-MSCs組(235.83±18.95、203.67±11.50和206.33±11.86;q=8.828,P＜0.01;q=3.802,P＜0.05;q=4.219,P＜0.05).LV-ARTN-MSCs組大鼠黑質TH暘性細胞百分比(64.05％±5.49％)顯著高于帕金森病組(34.18％±3.35％)、MSCs組(52.59％±4.48％)和LV-MSCs組(50.57％±4.41％;q=13.280、5.135、6.028,均P＜0.01),黑質TH蛋白錶達也明顯提高,同時慢病毒介導ARTN脩飾的MSCs可以在帕金森病大鼠腦內存活至少6週,主要集中在移植側的紋狀體區.細胞移植8週後,MSCs組(2.34±0.54)、LV-MSCs組(2.28±0.45)、LV-ARTN-MSCs組(6.54±0.51)與帕金森病組(0.87±0.07)大鼠相比,紋狀體多巴胺錶達明顯提高(q =5.233,P＜0.05;q=5.020,P＜0.01;q=20.190,P＜0.01),以LV-ARTN-MSCs組最明顯.結論 紋狀體移植慢病毒介導ARTN基因脩飾的MSCs對帕金森病模型大鼠有一定的治療作用.
목적 탐토만병독개도적artemin(ARTN)기인수식적골수간충질간세포(MSCs)대파금삼병모형대서잠재성치료작용급대뇌내상관단백표체적영향.방법 분리배양대서MSCs,장휴대ARTN적만병독재체전염MSCs;6-간다파알(6-OHDA)제비파금삼병대서모형,안수궤수자표법수궤분위가수술(Sham)조、파금삼병조、이식MSCs(MSCs)조、이식공병독전염적MSCs(LV-MSCs)조、이식만병독개도적ARTN기인수식적MSCs (LV-ARTN-MSCs)조,대파금삼병대서좌측문상체진행세포이식;술후2、4、6、8주,복강주사아복마배,진행행위학검측;채용Western인적급면역형광법검측각조대서흑질락안산간화매(TH)표체변화,이식ARTN수식적MSCs재대서뇌조직내표체;고효액상색보의검측각조대서문상체내다파알、이간기분을산、고향초산함량.결과 LV-ARTN-MSCs조재아복마배유발하30 min내선전권수(179.33±18.74)명현저우파금삼병조、MSCs조화LV-MSCs조(235.83±18.95、203.67±11.50화206.33±11.86;q=8.828,P＜0.01;q=3.802,P＜0.05;q=4.219,P＜0.05).LV-ARTN-MSCs조대서흑질TH양성세포백분비(64.05％±5.49％)현저고우파금삼병조(34.18％±3.35％)、MSCs조(52.59％±4.48％)화LV-MSCs조(50.57％±4.41％;q=13.280、5.135、6.028,균P＜0.01),흑질TH단백표체야명현제고,동시만병독개도ARTN수식적MSCs가이재파금삼병대서뇌내존활지소6주,주요집중재이식측적문상체구.세포이식8주후,MSCs조(2.34±0.54)、LV-MSCs조(2.28±0.45)、LV-ARTN-MSCs조(6.54±0.51)여파금삼병조(0.87±0.07)대서상비,문상체다파알표체명현제고(q =5.233,P＜0.05;q=5.020,P＜0.01;q=20.190,P＜0.01),이LV-ARTN-MSCs조최명현.결론 문상체이식만병독개도ARTN기인수식적MSCs대파금삼병모형대서유일정적치료작용.
Objective To investigate the potential therapeutic efficacy of lentivirus-mediated artemin (ARTN) gene modified bone marrow mesenchymal stem cells (MSCs) transplantation on the rat model of Parkinson' s disease (PD) and the effects on expression of brain-related proteins.Methods MSCs were isolated and cultured in vitro,transfected by recombinant lentiviral vectors carrying ARTN gene.The PD rat model established by 6-hydroxydopamine (6-OHDA) was randomly divided into 5 groups:Sham group,PD group,MSCs group,MSCs transfected with empty lentiviral vectors transplanted (LV-MSCs)group and MSCs transfected with recombinant lentiviral vectors carrying ARTN gene transplanted (LVARTN-MSCs) group.The MSCs,LV-MSCs and LV-ARTN-MSCs groups were transplanted into the left striatum of each rat model of PD and ethology tests in every group were made with intraperitoneal injection of apomorphine (APO) 2,4,6,8 weeks after transplantation.The expression of tyrosine hydroxylase (TH) protein in substantia nigra (SN) was measured by Western blotting and immunohistochemistry,and immunofluorescence showed ARTN gene modified MSCs expression in rat brain tissue.The levels of dopamine (DA),dihydroxy-phenylacetic acid and homovanillic acid in striatum of each group were detected by high performance liquid chromatography.Results After injection of APO,rotation frequency decreased in LV-ARTN-MSCs group,i.e.(179.33 ± 10.74) circles/30 min vs (235.83 ± 18.95),(203.67 ±11.50) and (206.33 ± 11.86) circles/30 min in PD,MSCs and LV-MSCs groups (q =8.828,P ＜ 0.01;q =3.802,P ＜ 0.05;q =4.219,P ＜ 0.05).The percentage of TH-positive cells in SN after cell transplantation was increased significantly in LV-ARTN-MSCs group (64.05％ ± 5.49％) when compared with PD group (34.18％ ±3.35％),MSCs group (52.59％ ±4.48％) and LV-MSCs group (50.57％ ± 4.41％),respectively (q =13.280,5.135,6.028,all P ＜0.01).At the same time,TH protein in SN after cell transplantation was also increased obviously in LV-ARTN-MSCs group.ARTN gene modified MSCs can survive for at least 6 weeks in the rat brain of PD,mainly concentrated in the transplantation side of striatum.Eight weeks later,the levels of DA in striatum after cell transplantation were elevated significantly in MSCs group (2.34 ± 0.54),LV-MSCs group (2.28 ± 0.45) and LV-ARTN-MSCs group (2.28 ± 0.45)when compared with PD group (0.87 ± 0.07) (q =5.233,P ＜ 0.05;q =5.020,P ＜ 0.01;q =20.190,P ＜ 0.01),and LV-ARTN-MSCs group showed the most significant improvement.Conclusion ARTN gene modified bone marrow MSCs transplanted into the striatum of brain may have therapeutic effects on rat models of PD.
