中华眼底病杂志
中華眼底病雜誌
중화안저병잡지
Chinese Journal of Ocular Fundus Diseases
2015年
6期
575-580
,共6页
王颖%江枫%陈庆中%张竹红%孟祥达%颜华
王穎%江楓%陳慶中%張竹紅%孟祥達%顏華
왕영%강풍%진경중%장죽홍%맹상체%안화
糖尿病视网膜病变/病理生理学%白细胞介素10%干细胞%动物实验
糖尿病視網膜病變/病理生理學%白細胞介素10%榦細胞%動物實驗
당뇨병시망막병변/병리생이학%백세포개소10%간세포%동물실험
Diabetic retinopathy/physiopathology%Interleukin-10%Stem cells%Animal experimentation
目的 观察白介素(IL)10修饰的内皮祖细胞(EPC)对糖尿病大鼠视网膜病变的影响.方法 采用密度梯度离心法从大鼠骨髓中提取并培养EPC,利用免疫荧光细胞化学染色法鉴定.取生长状态良好的EPC,每孔加入10μl慢病毒(LV)包装的IL10-绿色荧光蛋白(GFP)质粒(EPC-LV-IL10-GFP组)或LV包装的空白载体GFP质粒(EPC-LV-NC-GFP组),不加入任何质粒者作为EPC组.采用酶联免疫吸附测定法(ELISA)测量3组细胞培养基中肿瘤坏死因子(TNF)-α、IL10、IL8和血管内皮生长因子(VEGF)的表达.雄性Wistar大鼠168只分为正常对照组、糖尿病对照组、实验对照组及实验治疗组,分别为28、28、56、56只.后3组大鼠腹腔注射链脲佐菌素溶液建立糖尿病模型.建模成功后3个月,实验对照组、实验治疗组大鼠分别尾静脉注射EPC-LV-NC-GFP、EPC-LV-IL10-GFP;正常对照组、糖尿病对照组大鼠不给予任何干预.采用免疫组织化学染色法观察大鼠视网膜中GFP的表达;视网膜荧光染色铺片和伊凡思蓝(EB)定量检测大鼠血视网膜屏障的破坏程度;透射电子显微镜观察大鼠视网膜病理组织学的变化;逆转录聚合酶链反应(RT-PCR)检测大鼠视网膜组织中VEGF、金属蛋白酶(MMP)-9、血管生成素(Ang)-1、一氧化氮合酶(eNOS)、诱导型一氧化氮合酶(iNOS) mRNA的表达.结果 ELISA检测结果显示,与EPC组、EPC LV-NC GFP组比较,EPC-LV-IL10-GFP组细胞培养基中TNF-α、IL8浓度明显下降(F=28.910、14.242,P＜0.05);IL10、VEGF浓度明显升高(F=10.795、26.097,P＜0.05).免疫组织化学染色结果显示,正常对照组、糖尿病对照组大鼠视网膜组织GFP表达呈阴性;实验对照组、实验治疗组大鼠视网膜组织GFP表达呈阳性,主要表达在神经节细胞层、内核层及外丛状层.组间大鼠视网膜血管病理改变及EB平均渗漏量比较,糖尿病对照组较正常对照组明显加重(P＜o.05),实验对照组及实验治疗组较糖尿病对照组明显减轻(P＜0.05),干预后2、4周实验治疗组较实验对照组明显减轻(P＜0.05).与正常对照组比较,糖尿病对照组大鼠视网膜VEGF、MMP-9、Ang-1、iNOS mRNA表达明显提高(P＜0.05),eNOS mRNA表达明显降低(P＜0.05).与糖尿病对照组比较,实验对照组、实验治疗组VEGF、iNOS mRNA表达明显降低,且以实验治疗组降低更为明显(P＜0.05),Ang-1、MMP-9 mRNA表达无明显变化(P＞0.05),eNOSmRNA表达明显提高,且以实验治疗组的提高最明显(P＜0.05).结论 IL10修饰的EPC通过改善视网膜炎性微环境可减轻糖尿病大鼠视网膜病变程度;外源性EPC植入可能弥补糖尿病视网膜病变发生后EPC数量相对不足的缺陷,加强治疗效果.
目的 觀察白介素(IL)10脩飾的內皮祖細胞(EPC)對糖尿病大鼠視網膜病變的影響.方法 採用密度梯度離心法從大鼠骨髓中提取併培養EPC,利用免疫熒光細胞化學染色法鑒定.取生長狀態良好的EPC,每孔加入10μl慢病毒(LV)包裝的IL10-綠色熒光蛋白(GFP)質粒(EPC-LV-IL10-GFP組)或LV包裝的空白載體GFP質粒(EPC-LV-NC-GFP組),不加入任何質粒者作為EPC組.採用酶聯免疫吸附測定法(ELISA)測量3組細胞培養基中腫瘤壞死因子(TNF)-α、IL10、IL8和血管內皮生長因子(VEGF)的錶達.雄性Wistar大鼠168隻分為正常對照組、糖尿病對照組、實驗對照組及實驗治療組,分彆為28、28、56、56隻.後3組大鼠腹腔註射鏈脲佐菌素溶液建立糖尿病模型.建模成功後3箇月,實驗對照組、實驗治療組大鼠分彆尾靜脈註射EPC-LV-NC-GFP、EPC-LV-IL10-GFP;正常對照組、糖尿病對照組大鼠不給予任何榦預.採用免疫組織化學染色法觀察大鼠視網膜中GFP的錶達;視網膜熒光染色鋪片和伊凡思藍(EB)定量檢測大鼠血視網膜屏障的破壞程度;透射電子顯微鏡觀察大鼠視網膜病理組織學的變化;逆轉錄聚閤酶鏈反應(RT-PCR)檢測大鼠視網膜組織中VEGF、金屬蛋白酶(MMP)-9、血管生成素(Ang)-1、一氧化氮閤酶(eNOS)、誘導型一氧化氮閤酶(iNOS) mRNA的錶達.結果 ELISA檢測結果顯示,與EPC組、EPC LV-NC GFP組比較,EPC-LV-IL10-GFP組細胞培養基中TNF-α、IL8濃度明顯下降(F=28.910、14.242,P＜0.05);IL10、VEGF濃度明顯升高(F=10.795、26.097,P＜0.05).免疫組織化學染色結果顯示,正常對照組、糖尿病對照組大鼠視網膜組織GFP錶達呈陰性;實驗對照組、實驗治療組大鼠視網膜組織GFP錶達呈暘性,主要錶達在神經節細胞層、內覈層及外叢狀層.組間大鼠視網膜血管病理改變及EB平均滲漏量比較,糖尿病對照組較正常對照組明顯加重(P＜o.05),實驗對照組及實驗治療組較糖尿病對照組明顯減輕(P＜0.05),榦預後2、4週實驗治療組較實驗對照組明顯減輕(P＜0.05).與正常對照組比較,糖尿病對照組大鼠視網膜VEGF、MMP-9、Ang-1、iNOS mRNA錶達明顯提高(P＜0.05),eNOS mRNA錶達明顯降低(P＜0.05).與糖尿病對照組比較,實驗對照組、實驗治療組VEGF、iNOS mRNA錶達明顯降低,且以實驗治療組降低更為明顯(P＜0.05),Ang-1、MMP-9 mRNA錶達無明顯變化(P＞0.05),eNOSmRNA錶達明顯提高,且以實驗治療組的提高最明顯(P＜0.05).結論 IL10脩飾的EPC通過改善視網膜炎性微環境可減輕糖尿病大鼠視網膜病變程度;外源性EPC植入可能瀰補糖尿病視網膜病變髮生後EPC數量相對不足的缺陷,加彊治療效果.
목적 관찰백개소(IL)10수식적내피조세포(EPC)대당뇨병대서시망막병변적영향.방법 채용밀도제도리심법종대서골수중제취병배양EPC,이용면역형광세포화학염색법감정.취생장상태량호적EPC,매공가입10μl만병독(LV)포장적IL10-록색형광단백(GFP)질립(EPC-LV-IL10-GFP조)혹LV포장적공백재체GFP질립(EPC-LV-NC-GFP조),불가입임하질립자작위EPC조.채용매련면역흡부측정법(ELISA)측량3조세포배양기중종류배사인자(TNF)-α、IL10、IL8화혈관내피생장인자(VEGF)적표체.웅성Wistar대서168지분위정상대조조、당뇨병대조조、실험대조조급실험치료조,분별위28、28、56、56지.후3조대서복강주사련뇨좌균소용액건립당뇨병모형.건모성공후3개월,실험대조조、실험치료조대서분별미정맥주사EPC-LV-NC-GFP、EPC-LV-IL10-GFP;정상대조조、당뇨병대조조대서불급여임하간예.채용면역조직화학염색법관찰대서시망막중GFP적표체;시망막형광염색포편화이범사람(EB)정량검측대서혈시망막병장적파배정도;투사전자현미경관찰대서시망막병리조직학적변화;역전록취합매련반응(RT-PCR)검측대서시망막조직중VEGF、금속단백매(MMP)-9、혈관생성소(Ang)-1、일양화담합매(eNOS)、유도형일양화담합매(iNOS) mRNA적표체.결과 ELISA검측결과현시,여EPC조、EPC LV-NC GFP조비교,EPC-LV-IL10-GFP조세포배양기중TNF-α、IL8농도명현하강(F=28.910、14.242,P＜0.05);IL10、VEGF농도명현승고(F=10.795、26.097,P＜0.05).면역조직화학염색결과현시,정상대조조、당뇨병대조조대서시망막조직GFP표체정음성;실험대조조、실험치료조대서시망막조직GFP표체정양성,주요표체재신경절세포층、내핵층급외총상층.조간대서시망막혈관병리개변급EB평균삼루량비교,당뇨병대조조교정상대조조명현가중(P＜o.05),실험대조조급실험치료조교당뇨병대조조명현감경(P＜0.05),간예후2、4주실험치료조교실험대조조명현감경(P＜0.05).여정상대조조비교,당뇨병대조조대서시망막VEGF、MMP-9、Ang-1、iNOS mRNA표체명현제고(P＜0.05),eNOS mRNA표체명현강저(P＜0.05).여당뇨병대조조비교,실험대조조、실험치료조VEGF、iNOS mRNA표체명현강저,차이실험치료조강저경위명현(P＜0.05),Ang-1、MMP-9 mRNA표체무명현변화(P＞0.05),eNOSmRNA표체명현제고,차이실험치료조적제고최명현(P＜0.05).결론 IL10수식적EPC통과개선시망막염성미배경가감경당뇨병대서시망막병변정도;외원성EPC식입가능미보당뇨병시망막병변발생후EPC수량상대불족적결함,가강치료효과.
Objective To observe the effect of interleukin (IL) 10 modified endothelial progenitor cells (EPC) in diabetic retinopathy (DR).Methods EPC cells were collected and cultivated from the bone marrow of rats and identified by immuno-fluorescence staining.EPC cells were infected with lentivirus (LV) of EPC-LV-IL10-GFP (EPC-LV-IL10-GFP group) or EPC-LV-NC-GFP (GFP group).EPC cells without lentivirus infection was the EPC group.Enzyme-linked immuno sorbent assay (ELISA) was used to measure the concentrations of tumor necrosis factor (TNF)-α, IL10, IL8 and vascular endothelial growth factor (VEGF) in the supernatant of these three groups.168 male Wistar rats were divided into normal control group (28 rats), diabetes mellitus (DM) group (28 rats), DM-blank control group (56 rats) and DM-intervention group (56 rats).DM was introduced in the latter 3 groups by streptozotocin intravenous injection.Three months later, the rats in the DM-blank control group and DM intervention group were injected with EPC-LV-NC-GFP or EPC-LV-IL10-GFP by tail vein, respectively.Immunohistochemistry was used to observe the GFP expression in rat retinas.The blood-retinal barrier breakdown was detected by Evans blue (EB) dye.The retinal histopathologic changes were observed by transmission electron microscope.The mRNA level of VEGF, matrix metallproteinases-9 (MMP 9), angiopoietin-1 (Ang 1),inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) in retina were measured by reverse transcription-polymerase chain reaction (RT-PCR).Results ELISA showed that the levels of TNF-α and IL8 in the supernatant significantly decreased , while the levels of IL10 and VEGF increased (P＜0.05) in EPC LV-IL10 GFP group.GFP expressed in the retina of blank control group and intervention group, mainly in the ganglion cell layer, inner nuclear layer and outer plexiform layer.The retinal blood vessel pathological change and EB permeability significantly decreased in intervention group compared with DM group (P ＜0.05), and blank control group (P＜ 0.05).RT-PCR revealed that the mRNA level of VEGF, MMP-9 and Ang-1 significantly increased, and eNOS decreased in DM group compared to the normal control group (P＜0.05).The mRNA level of VEGF and iNOS decreased, eNOS increased while Ang 1 and MMP-9 had not changed in DM-blank control group and DM-intervention group compared with DM group (P ＜ 0.05).Conclusions IL10 modified EPC can improve the inflammative microenvironment and suppressed the pathogenesis of DR.Furthermore, EPC transplantation can increase the number of EPC and exerted their effect.
