不同劑量的重組腺相關(guān)病毒載體介導(dǎo)的人血管抑素對(duì)糖尿病腎病大鼠

1、不同劑量的重組腺相關(guān)病毒載體介導(dǎo)的人血管抑素對(duì)糖尿病腎病大鼠腎功能的影響         10-10-30 16:18:00     作者：李偉，應(yīng)長江，林紅曉    編輯：studa20【摘要】  目的 探討不同劑量的重組腺相關(guān)病毒載體介導(dǎo)的人血管抑素(recombinant adeno-associated virus carrying human angiostatin gene, rAAV-AS)對(duì)糖尿病腎病(diab

2、etic nephropathy, DN)大鼠腎功能的影響。方法 60只清潔級(jí)雄性SD大鼠隨機(jī)抽取10只作為正常對(duì)照組(NC組)，其余造模為糖尿病大鼠。大鼠空腹12 h腹腔注射鏈尿佐菌素(STZ)60 mg/kg，72 h后尾靜脈測血糖大于16.7 mmol/L記為造模成功，適應(yīng)性喂養(yǎng)1周后，把糖尿病大鼠隨機(jī)分為DN組(n=10)，rAAV-0組(n=10), AS1組(n=10, 1×1012 VG/kg)，AS2組(n=10, 2×1012 VG/kg)，AS3組(n=10, 4×1012 VG/kg)。AS1組、AS2組和AS3組3組大鼠尾靜脈注射rAAV-

3、AS, NC組和DN組分別注射同等劑量的生理鹽水，rAAV-0組注射同等劑量rAAV-0空載體;從第2周起，整個(gè)實(shí)驗(yàn)過程共持續(xù)8周。8周后檢測各組大鼠的體重(body weight, BW)、腎重(kidney weight, KW)，計(jì)算腎指數(shù)(kidney index, KI)，檢測血糖(blood glucose, BG)、糖化血紅蛋白(hemoglobin A1c, HbA1c)、尿素氮(blood urea nitrogen, BUN)、血肌酐(serum creatinine, SCr)、尿微量白蛋白(miscroalbunminuria, MAU)。結(jié)果 與NC組相比，DN、rA

4、AV-0、AS1組的KI、MAU、BUN、SCr、BG、HbA1c明顯增高(P<0.05);與DN組相比，AS2組和AS3組KI、MAU、BUN、SCr明顯降低(P<0.05);與rAAV-0組相比，AS2組和AS3組KI、MAU、BUN、SCr降低(P<0.05);與AS1組相比，AS2組和AS3組KI、MAU、BUN、SCr降低(P<0.05)。結(jié)論 中等劑量和大劑量 rAAV-AS對(duì)DN大鼠腎臟具有保護(hù)作用，且不依賴血糖降低而起作用。 【關(guān)鍵詞】  糖尿病腎病;腺相關(guān)病毒載體介導(dǎo)的血管抑素;基因治療Abstract:Objective To invest

5、igate the effect of different doses of recombinant adeno-associated virus carrying human angiostatin gene (rAAV-AS) on the renal function in rats with diabetic nephropathy. Methods 10 out of 60 male SD rats of clean grade were randomly allotted as the normal control group, while the others were used

6、 to establish models of diabetic rats. Streptozotocin (STZ) 60 mg/kg was intraperitoneally injected in rats at the end of 12 h of fasting to induce diabetic model. 72 h later, the blood glucose level of caudal vein was above 16.7 mmol/L, and the diabetic model was marked as successful. After a week

7、of adaptation, diabetic rats were randomized into 5 groups (n=10): group DN, group rAAV-0, group AS1 (1×1012 VG/kg), group AS2 (2×1012 VG/kg), group AS3 (4×1012 VG/kg). Different doses of rAAV-AS were injected into groups AS1, AS2 and AS3 from the caudal vein, and the corresponding do

8、ses of normal saline were injected in group NC and corresponding doses of rAAV-0 idler injected in group DN. At the end of the 8th weeks, their body weight (BW) and kidney weight (KW) were measured to calculate kidney index (KI). The blood glucose (BG), glycosylated hemoglobin (HbA1c), miscroalbunmi

9、nuria (MAU), blood urea nitrogen (BUN) and serum creatinine (SCr) were determined.Results Compared with group NC, the levels of KI, BG, HbA1c, MAU, BUN and SCr increased significantly in DN, rAAV-0 and AS1 groups (P<0.05). Compared with group DN, the levels of KI, MAU, BUN and SCr significantly d

10、ecreased in groups AS2 and AS3 (P<0.05). Compared with group rAAV-0, the levels of KI, MAU, BUN and SCr significantly decreased in groups AS2 and AS3 (P<0.05). Compared with group AS1, the levels of KI, MAU, BUN and SCr significantly decreased in groups AS2 and AS3 (P<0.05).Conclusions rAAV

11、-AS of medium and large doses has protective effect on the kidneys in diabetic rats, independent of the decrease of blood glucose level.Key words： diabetic nephropathy; recombinant adeno-associated virus carrying human angiostatin gene; gene therapy糖尿病腎病(diabetic nephropathy， DN)是糖尿病最常見的慢性微血管并發(fā)癥之一,在

12、1型糖尿病中發(fā)病率約33%44%,在2型糖尿病中約15%60%,并是造成終末期腎病(end-stage renal disease，ESRD)的重要原因之一。但目前臨床應(yīng)用的藥物尚不能有效地逆轉(zhuǎn)DN的進(jìn)程，因此找到一種可靠的方法預(yù)防和治療糖尿病腎病迫在眉睫。血管抑素(angiostatin, AS)具有抑制血管生成、抗纖維化等生物學(xué)活性，研究表明AS能降低DN的蛋白尿1，但是由于注射AS在體內(nèi)活性不穩(wěn)定、表達(dá)效率差，因此我們使用不同劑量重組腺相關(guān)病毒載體介導(dǎo)的人血管抑素(recombinant adeno-associated virus carrying human angiostatin

13、gene, rAAV-AS)治療DN大鼠，觀察其對(duì)DN大鼠尿微量蛋白和腎功能的影響，以明確不同劑量rAAV-AS對(duì)早期DN大鼠的作用。1 材料和方法1.1 實(shí)驗(yàn)動(dòng)物和試劑 雄性SD大鼠，體重160180 g,8周齡，由徐州醫(yī)學(xué)院實(shí)驗(yàn)動(dòng)物中心提供。鏈脲佐菌素(STZ)購自美國Sigma公司,rAAV-AS由本元正陽公司構(gòu)建。1.2 實(shí)驗(yàn)分組和模型制備 60只清潔級(jí)雄性SD大鼠適應(yīng)性喂養(yǎng)標(biāo)準(zhǔn)大鼠飼料1周后，從中隨機(jī)抽取10只作為正常對(duì)照組(NC組)，其余造模為糖尿病大鼠。大鼠空腹12 h腹腔注射STZ 60 mg/kg，NC組僅注射檸檬酸-檸檬酸鈉緩沖液。72 h后尾靜脈測血糖大于16.7 mmo

14、l/L記為造模成功，適應(yīng)性喂養(yǎng)1周后，把糖尿病大鼠隨機(jī)分為DN組(n=10)，腺相關(guān)病毒空載體組(rAAV-0，n=10), rAAV-AS低劑量治療組(AS1組, n=10, 1×1012 VG/kg)， rAAV-AS中劑量治療組(AS2組, n=10, 2×1012 VG/kg)，rAAV-AS高劑量治療組(AS3組, n=10, 4×1012 VG/kg)。rAAV-AS各劑量組大鼠尾靜脈注射rAAV-AS, NC組和DN組分別注射同等劑量的生理鹽水，rAAV-0組注射同等劑量rAAV-0空載體;從第2周起，整個(gè)實(shí)驗(yàn)過程共持續(xù)8周。1.3 標(biāo)本收集 各組大鼠于8周末測量體重(BW)，尾靜脈取血測定血糖，用代謝籠收集24 h尿液、離心，保存于-40冰箱中，以待測尿白蛋白。大鼠隔夜空腹，1%的戊巴比妥鈉(50 mg/kg 腹腔注射)麻醉下心臟采血、離心、取上清，-40冰箱保存，以備測定糖化血紅蛋白(HbA1c)、尿素氮(BUN)、血肌酐(SCr)。然后，取大鼠