中华老年医学杂志
中華老年醫學雜誌
중화노년의학잡지
Chinese Journal of Geriatrics
2014年
6期
648-652
,共5页
倪炯%王培军%王国良%梁挺
倪炯%王培軍%王國良%樑挺
예형%왕배군%왕국량%량정
阿尔茨海默病%小神经胶质细胞%RNA,小分子干扰
阿爾茨海默病%小神經膠質細胞%RNA,小分子榦擾
아이자해묵병%소신경효질세포%RNA,소분자간우
Alzheimer disease%Microglia%RNA,small interfering
目的 观察RNA干扰(RNAi)沉默小胶质细胞内的P2X7受体(P2X7R)对小胶质细胞释放炎性介质白细胞介素1β(IL-1β)、肿瘤坏死因子α(TNF-α)和一氧化氮(NO)的影响. 方法 构建靶向P2X7R基因的小干扰RNA(siRNA).以脂质体为转染剂将siRNA转染到经β淀粉样蛋白(Aβ1-42)激活的原代培养的小胶质细胞内.根据刺激物不同分为Aβ1-42、Aβ1-42/siNC、Aβ1-42/siP2X7R 及空白对照组.处理48 h后,收集各组小胶质细胞,用CCK-8检测细胞存活率,用Real-Time PCR和Western blot检测P2X7R的mRNA和蛋白表达水平,用免疫细胞化学染色观察细胞形态及P2X7R免疫反应强度;收集各组细胞上清液测定IL-1β、TNF-α和NO浓度. 结果 Aβ1-42和siRNA对小胶质细胞存活率没有影响,与空白对照组比较差异无统计学意义.RNA干扰后Aβ1-42/siP2X7R组P2X7R的mRNA和蛋白表达均显著下降,免疫细胞化学染色该组小胶质细胞活性和P2X7R免疫反应强度均降低.沉默P2X7R后,Aβ1-42、Aβ1-42/siNC、Aβ1-42/siP2X7R及空白对照组细胞上清液IL-1β浓度分别为(52.54±3.21)μg/L、(54.94±2.54) μg/L、(28.70±3.58)μg/L和(24.55±4.34) μg/L;TNF-α浓度分别为(64.40±4.80)μg/L、(60.94±1.63)μg/L、(25.69±3.13)μg/L和(19.21±1.97)μg/L.Aβ1-42/siP2X7R组细胞上清液IL-1β及TNF-α浓度均低于Aβ1-42和Aβ1-42/siNC组(P＜0.05),与空白对照组比较差异无统计学意义.Aβ1-42/siP2X7R组细胞上清液NO浓度为(1.33±0.19)μmol/L,低于Aβ1-42组(4.49±0.28) μmol/L和Aβ1-42/siNC组(4.78±0.33) μmol/L,与空白对照组(1.07±0.36) μmol/L比较差异无统计学意义. 结论 RNA干扰可有效沉默小胶质细胞内的P2X7R,抑制小胶质细胞释放炎性介质IL-1β、TNF-α和NO.P2X7R有望成为RNA干扰治疗阿尔茨海默病的一个新靶点.
目的 觀察RNA榦擾(RNAi)沉默小膠質細胞內的P2X7受體(P2X7R)對小膠質細胞釋放炎性介質白細胞介素1β(IL-1β)、腫瘤壞死因子α(TNF-α)和一氧化氮(NO)的影響. 方法 構建靶嚮P2X7R基因的小榦擾RNA(siRNA).以脂質體為轉染劑將siRNA轉染到經β澱粉樣蛋白(Aβ1-42)激活的原代培養的小膠質細胞內.根據刺激物不同分為Aβ1-42、Aβ1-42/siNC、Aβ1-42/siP2X7R 及空白對照組.處理48 h後,收集各組小膠質細胞,用CCK-8檢測細胞存活率,用Real-Time PCR和Western blot檢測P2X7R的mRNA和蛋白錶達水平,用免疫細胞化學染色觀察細胞形態及P2X7R免疫反應彊度;收集各組細胞上清液測定IL-1β、TNF-α和NO濃度. 結果 Aβ1-42和siRNA對小膠質細胞存活率沒有影響,與空白對照組比較差異無統計學意義.RNA榦擾後Aβ1-42/siP2X7R組P2X7R的mRNA和蛋白錶達均顯著下降,免疫細胞化學染色該組小膠質細胞活性和P2X7R免疫反應彊度均降低.沉默P2X7R後,Aβ1-42、Aβ1-42/siNC、Aβ1-42/siP2X7R及空白對照組細胞上清液IL-1β濃度分彆為(52.54±3.21)μg/L、(54.94±2.54) μg/L、(28.70±3.58)μg/L和(24.55±4.34) μg/L;TNF-α濃度分彆為(64.40±4.80)μg/L、(60.94±1.63)μg/L、(25.69±3.13)μg/L和(19.21±1.97)μg/L.Aβ1-42/siP2X7R組細胞上清液IL-1β及TNF-α濃度均低于Aβ1-42和Aβ1-42/siNC組(P＜0.05),與空白對照組比較差異無統計學意義.Aβ1-42/siP2X7R組細胞上清液NO濃度為(1.33±0.19)μmol/L,低于Aβ1-42組(4.49±0.28) μmol/L和Aβ1-42/siNC組(4.78±0.33) μmol/L,與空白對照組(1.07±0.36) μmol/L比較差異無統計學意義. 結論 RNA榦擾可有效沉默小膠質細胞內的P2X7R,抑製小膠質細胞釋放炎性介質IL-1β、TNF-α和NO.P2X7R有望成為RNA榦擾治療阿爾茨海默病的一箇新靶點.
목적 관찰RNA간우(RNAi)침묵소효질세포내적P2X7수체(P2X7R)대소효질세포석방염성개질백세포개소1β(IL-1β)、종류배사인자α(TNF-α)화일양화담(NO)적영향. 방법 구건파향P2X7R기인적소간우RNA(siRNA).이지질체위전염제장siRNA전염도경β정분양단백(Aβ1-42)격활적원대배양적소효질세포내.근거자격물불동분위Aβ1-42、Aβ1-42/siNC、Aβ1-42/siP2X7R 급공백대조조.처리48 h후,수집각조소효질세포,용CCK-8검측세포존활솔,용Real-Time PCR화Western blot검측P2X7R적mRNA화단백표체수평,용면역세포화학염색관찰세포형태급P2X7R면역반응강도;수집각조세포상청액측정IL-1β、TNF-α화NO농도. 결과 Aβ1-42화siRNA대소효질세포존활솔몰유영향,여공백대조조비교차이무통계학의의.RNA간우후Aβ1-42/siP2X7R조P2X7R적mRNA화단백표체균현저하강,면역세포화학염색해조소효질세포활성화P2X7R면역반응강도균강저.침묵P2X7R후,Aβ1-42、Aβ1-42/siNC、Aβ1-42/siP2X7R급공백대조조세포상청액IL-1β농도분별위(52.54±3.21)μg/L、(54.94±2.54) μg/L、(28.70±3.58)μg/L화(24.55±4.34) μg/L;TNF-α농도분별위(64.40±4.80)μg/L、(60.94±1.63)μg/L、(25.69±3.13)μg/L화(19.21±1.97)μg/L.Aβ1-42/siP2X7R조세포상청액IL-1β급TNF-α농도균저우Aβ1-42화Aβ1-42/siNC조(P＜0.05),여공백대조조비교차이무통계학의의.Aβ1-42/siP2X7R조세포상청액NO농도위(1.33±0.19)μmol/L,저우Aβ1-42조(4.49±0.28) μmol/L화Aβ1-42/siNC조(4.78±0.33) μmol/L,여공백대조조(1.07±0.36) μmol/L비교차이무통계학의의. 결론 RNA간우가유효침묵소효질세포내적P2X7R,억제소효질세포석방염성개질IL-1β、TNF-α화NO.P2X7R유망성위RNA간우치료아이자해묵병적일개신파점.
Objective To observe the effect of silencing P2X7 receptor (P2X7R) by RNA interference on microglial releasing interleukin 1β (IL-1β),tumor necrosis factor-α (TNF-α) and nitric oxide (NO).Methods The small interfering RNA (siRNA) targeting P2X7R gene was observed.The primary microglial cells activated by amyloid-β (Aβ-42) were infected with the lipofectaminesiRNA.The groups were designated as Aβ-1-42,Aβ1-42/siNC,Aβ1-42/siP2X7R and blank control groups according to the different stimulus.After RNA interference for 48 hours,the microglial cells were collected.The survival rate of microglia was detected by CCK-8.The levels of P2X7R mRNA and protein were detected by Real-time PCR and Western blotting respectively.The microglial morphology and the P2X7R immune response were observed by immunocytochemistry staining.Then levels of IL-1β,TNF-α and NO in the supernatantwere measured.Results The Aβ1-42 and siRNA had no effects on the survival rate of microglia.After RNA interference silencing P2X7R,the expression levels of P2X7R mRNA and protein in Aβ1-42/siP2X7R group were decreased significantly,and in this group,the microglial activity and P2X7R immune response were all reduced.In Aβ1-42,Aβ1-42/siNC,Aβ1-42 /siP2X7R and blank control groups,the supernatant levels of IL-1β were (52.54±3.21) μg L,(54.94±2.54) μg/L,(28.70±3.58) μg/L,(24.55±4.34) μg/L,respectively and the supernatant levels of TNF-α were (64.40±4.80) μg/L,(60.94±1.63) μg/L,(25.69±3.13) μg/l,(19.21±1.97)μg/L,respectively.As compared with Aβ1-42 and Aβ-42/siNC groups,the levels of IL 1β and TNF-α in Aβ1-42 /siP2X7R group was decreased significantly (P＜ 0.05),and IL-1β level had no significant difference between Aβ1-42/siP2X7R and blank control groups.The same result was observed in the level of NOin the supernatant.The levels of NO were (1.33±0.19) μmol/L,(4.49±0.28) μmol/L,(4.78±0.33) μmol/L and (1.07±0.36) μmol/L in Aβ1-42/siP2X7R,Aβ1-42,Aβ1-42 /siNC and blank control groups respectively.Conclusions The silencing expression of P2X7 R by RNA interference effectively decreases the levels of IL-1β,TNF-α and NO released by microglia.P2X7 R can be used as an effective therapeutic target for RNA interference treatment of Alzheimer's disease.
