中华神经科杂志
中華神經科雜誌
중화신경과잡지
Chinese Journal of Neurology
2011年
10期
689-693
,共5页
栾兴华%洪道俊%乔晓会%吕鹤%王朝霞%袁云
欒興華%洪道俊%喬曉會%呂鶴%王朝霞%袁雲
란흥화%홍도준%교효회%려학%왕조하%원운
夏科-马里-图斯病%连接蛋白类%血管%内皮细胞
夏科-馬裏-圖斯病%連接蛋白類%血管%內皮細胞
하과-마리-도사병%련접단백류%혈관%내피세포
Charcot-Marie-Tooth disease%Connexins%Blood vessels%Endothelial cells
目的 观察突变缝隙连接蛋白32( Cx32)在X连锁的Charcot-Marie-Tooth病1型(CMTX1)患者血管内皮细胞的表达规律。方法 对3例经Cx32基因检查证实的CMTX1患者（突变位点分别为c.379A＞T、c.533A＞G和c.590C＞T点突变）进行腓肠神经活体组织检查，同时取非CMTXI患者的神经作为对照。以第Ⅷ因子、缝隙连接蛋白40和Cx32单克隆抗体为一抗，对患者及对照者标本中的神经滋养血管进行免疫荧光标记。同时构建携带上述3个点突变的以增强型绿色荧光蛋白(enhanced green fluorescence protein，EGFP)为报告基因的重组质粒pEGFP-N1-CX32质粒，转染至HeLa细胞，并进行Cx32和内质网标志蛋白葡萄糖调节蛋白78免疫荧光染色，以观察Cx32蛋白在HeLa细胞的分布规律。结果 Cx32蛋白呈一定间隔规律性点状表达于对照组血管内皮细胞间的缝隙连接部位，与Cx40蛋白共表达；3例Cx32基因突变患者的血管内皮细胞膜的Cx32蛋白表达显著减少。细胞转染显示c.379A＞T突变的Cx32蛋白主要以团块状蓄积在HeLa细胞的胞质内，c.533A＞G突变的Cx32蛋白仅有少量表达于核周，c.590C＞T突变的Cx32蛋白在胞膜上和胞质内均有点状分布，较野生型显著减少。3种突变的Cx32蛋白在HeLa细胞内的分布和内质网标记不重叠。结论 不同Cx32基因突变均可导致CMTX1的血管内皮细胞膜Cx32蛋白表达下降，该蛋白主要聚集在细胞的内质网外。
目的 觀察突變縫隙連接蛋白32( Cx32)在X連鎖的Charcot-Marie-Tooth病1型(CMTX1)患者血管內皮細胞的錶達規律。方法 對3例經Cx32基因檢查證實的CMTX1患者（突變位點分彆為c.379A＞T、c.533A＞G和c.590C＞T點突變）進行腓腸神經活體組織檢查，同時取非CMTXI患者的神經作為對照。以第Ⅷ因子、縫隙連接蛋白40和Cx32單剋隆抗體為一抗，對患者及對照者標本中的神經滋養血管進行免疫熒光標記。同時構建攜帶上述3箇點突變的以增彊型綠色熒光蛋白(enhanced green fluorescence protein，EGFP)為報告基因的重組質粒pEGFP-N1-CX32質粒，轉染至HeLa細胞，併進行Cx32和內質網標誌蛋白葡萄糖調節蛋白78免疫熒光染色，以觀察Cx32蛋白在HeLa細胞的分佈規律。結果 Cx32蛋白呈一定間隔規律性點狀錶達于對照組血管內皮細胞間的縫隙連接部位，與Cx40蛋白共錶達；3例Cx32基因突變患者的血管內皮細胞膜的Cx32蛋白錶達顯著減少。細胞轉染顯示c.379A＞T突變的Cx32蛋白主要以糰塊狀蓄積在HeLa細胞的胞質內，c.533A＞G突變的Cx32蛋白僅有少量錶達于覈週，c.590C＞T突變的Cx32蛋白在胞膜上和胞質內均有點狀分佈，較野生型顯著減少。3種突變的Cx32蛋白在HeLa細胞內的分佈和內質網標記不重疊。結論 不同Cx32基因突變均可導緻CMTX1的血管內皮細胞膜Cx32蛋白錶達下降，該蛋白主要聚集在細胞的內質網外。
목적 관찰돌변봉극련접단백32( Cx32)재X련쇄적Charcot-Marie-Tooth병1형(CMTX1)환자혈관내피세포적표체규률。방법 대3례경Cx32기인검사증실적CMTX1환자（돌변위점분별위c.379A＞T、c.533A＞G화c.590C＞T점돌변）진행비장신경활체조직검사，동시취비CMTXI환자적신경작위대조。이제Ⅷ인자、봉극련접단백40화Cx32단극륭항체위일항，대환자급대조자표본중적신경자양혈관진행면역형광표기。동시구건휴대상술3개점돌변적이증강형록색형광단백(enhanced green fluorescence protein，EGFP)위보고기인적중조질립pEGFP-N1-CX32질립，전염지HeLa세포，병진행Cx32화내질망표지단백포도당조절단백78면역형광염색，이관찰Cx32단백재HeLa세포적분포규률。결과 Cx32단백정일정간격규률성점상표체우대조조혈관내피세포간적봉극련접부위，여Cx40단백공표체；3례Cx32기인돌변환자적혈관내피세포막적Cx32단백표체현저감소。세포전염현시c.379A＞T돌변적Cx32단백주요이단괴상축적재HeLa세포적포질내，c.533A＞G돌변적Cx32단백부유소량표체우핵주，c.590C＞T돌변적Cx32단백재포막상화포질내균유점상분포，교야생형현저감소。3충돌변적Cx32단백재HeLa세포내적분포화내질망표기불중첩。결론 불동Cx32기인돌변균가도치CMTX1적혈관내피세포막Cx32단백표체하강，해단백주요취집재세포적내질망외。
Objective To investigate expression distribution of mutant connexin 32 (Cx32) protein in human endothelial cells in patients with X-linked Charcot-Marie-Tooth disease type 1 ( CMTX1 ) .Methods Nerve biopsies were performed in 3 patients with CMTX1 and in 3 non-CMTX1 controls. Cx32 mutations of c. 379A ＞ T( I127F), c. 533A ＞ G(D178G) and c. 590C ＞ T(A197V) were identified in these 3 patients respectively. Immunofluorescent (IMF) staining of nerve blood vessel was processed with antibodies against Cx32, Yon Willebrand factor and Cx40. The mutant Cx32 was constructed in pEGFP-N plasmid (pEGFP-N1-Cx32) and was transfected in HeLa cells. Cx32 and GRP78, a marker of endoplasmic reticulum ( ER), were stained by IMF in HeLa cells to investigate expression of mutant Cx32. Results In 3 control cases, Cx32 was visualized by IMF staining as dots along gap junction of vascular endothelial cells,and it was coexisted with Cx40. However, immunoreactivity of Cx32 in 3 patients was predominantly decreased and was not located in endothelial gap junction. The transfection of 3 Cx32 mutants into HeLa cells demonstrated the pathogenic changes. The cells with the mutation c. 379A ＞T found Cx32 accumulations in the cytoplasm; the cells with mutation c. 533A ＞G showed few staining positive dots surrounding the nuclear and the cells with c. 590C ＞ T showed dot-like expression of Cx32 both in the cytoplasmicand cell membrane. The mutant Cx32 was not overlapped with expression of the marker of ER.ConclusionsMutant Cx32 might cause dysfunction of endothelial gap-junctions due to the abnormal expression of Cx32 in level and location in the vascular endothelial cells of CMTX1 patients.
