【作者】 张新科；

【导师】 王凤山；

【作者基本信息】 山东大学 ， 微生物与生化药学， 2012， 博士

【摘要】 糖尿病视网膜病变、年龄相关性黄斑变性等疾病会引起眼底部位的血管增生,使视功能受到严重损害,甚至失明。目前,临床上大都采用眼内注射或激光光凝法治疗眼底血管增生,而这些方式往往会造成视网膜严重不可逆损伤。因此,寻找能够防治眼内血管生成且副作用小的药物具有重大的社会和经济意义。内皮抑素(endostatin, Es)是一种内源性血管内皮细胞增殖的强效抑制剂,我国已将重组人血管内皮抑素研制成具有自主知识产权的国家一类新药(Endostar,即恩度、YH16)用以治疗非小细胞肺癌。与传统化疗药物相比,Es具有毒副作用小、不易产生耐药性等优点。Es不仅在治疗肿瘤方面有其独特的优势,在防治眼部新生血管性疾病方面也取得一些可喜的成就。研究证明,通过球结膜下注射、玻璃体腔注射,Es及Es基因均可抑制眼部新生血管的形成。但是,由于Es是生物大分子,不能透过眼球屏障,必须通过眼内注射方式才能发挥疗效,而这种操作方式难度大,容易损伤眼球内组织,甚至会对病人造成不可逆的损伤,所以寻找简便、安全、有效的给药途径具有重要的临床意义。能够穿透细胞膜的HIV-1反式转录因子的蛋白转导域(protein transduction domain of the transacting activator of transcription, Tat PTD)能够携带穿透能力差的药物进入细胞,且具有携带药物透穿透眼球屏障的可能性。故本课题拟利用穿膜肽Tat PTD的穿膜作用和内皮抑素的抑制新生血管生成的作用,将二者通过基因工程的手段融合在一起,以期获得能够穿透眼球屏障的内皮抑素,达到通过简单的局部滴眼给药预防视网膜或脉络膜血管增生的目标。本课题的研究内容及取得的主要成果有以下几个方面。1Tat PTD-endostatin (Tat PTD-Es)在毕赤酵母中的表达及纯化本课题构建了Tat PTD-Es融合基因及酵母分泌型表达载体pGAPZαA/Tat PTD-Es(pG/TE)及pGAPZaA/Es(pG/E)。采用电转化法将重组质粒转入毕赤酵母菌(GS115),筛选阳性菌落,SDS-PAGE检测目的蛋白。利用镍离子亲和层析对表达产物进行初步纯化。结果表明Tat PTD-Es的毕赤酵母表达体系构建成功,但表达量低。2Tat PTD-Es在大肠杆菌中的表达、复性及纯化采用大肠杆菌表达系统对Tat PTD-Es及Es进行了融合表达。选用pET28a作为表达载体,构建重组质粒pET28a/Tat PTD-Es (pET28a/TE)和pET28a/Es (pET28a/E),转入E.coli Rosetta(DE3)中诱导表达目的蛋白。选用温度37℃,诱导时间4h的条件对工程菌株进行诱导,SDS-PAGE检测目的蛋白。摸索了包涵体的洗涤条件,采用直接稀释法对包涵体蛋白Tat PTD-Es及Es进行复性,采用Hi strap预装柱对复性后的蛋白进行了纯化,得到了高纯度的目的蛋白,SDS-PAGE结果显示Tat PTD-Es的分子量为22kD, Western blot结果显示目的蛋白可以与6xHis抗体结合,表明目的蛋白含有6xHis标签。3Tat PTD-Es的体内外活性及入胞机制研究采用CCK-8法测定了Tat PTD-Es及Es对人脐静脉内皮细胞EAHY926增殖的抑制作用,结果表明：Tat PTD-Es及Es均能显著地抑制内皮细胞的增殖,且抑制率呈现明显的剂量依赖性,即随着浓度的增加对细胞生长的抑制率也增加;Tat PTD-Es在低浓度时(0.2μmol/L,0.8μmol/L)活性要高于Es,当浓度大于4μmol/L时,Tat PTD-Es及Es抑制率均大于80%。建立鸡胚绒毛尿囊膜(CAM)模型,评价复性纯化后的Tat PTD-Es及Es对CAM血管生成的影响,以生理盐水作为空白对照,碱性成纤维细胞生长因子(bFGF)作为阴性对照,观察了Tat PTD-Es及Es对bFGF诱导的CAM血管生成的抑制作用。与bFGF组(12.18±4.72)比较,Tat PTD-Es及Es组的CAM血管数分别减少至(30.74±6.47)及(14.56±6.21)(p<0.05),结果表明,Tat PTD-Es及Es均能够明显抑制bFGF诱导的CAM血管的生成。采用荧光显微镜和流式细胞术对Tat PTD-Es与Es的入胞能力进行了比较。结果表明FITC标记的Tat PTD-Es与Es与人脐静脉内皮细胞EAHY926共孵育4h后,Es组的荧光信号很弱,细胞阳性率仅为33.61%；而Tat PTD-Es组的荧光信号与Es相比有很明显的增强,细胞阳性率高达99.28%,提示Tat PTD与Es融合表达后有利于蛋白质穿透细胞膜进入细胞,而使进入细胞的蛋白质大大增加,可能有利于其在胞内更好地发挥作用。考察了蛋白Tat PTD-Es浓度、孵育时间对人脐静脉内皮细胞EAHY926摄取蛋白质的影响及各种胞吞途径抑制剂对Tat PTD-Es进入人脐静脉内皮细胞EAHY926的影响。结果表明,蛋白质浓度在1μmol/L～10μmol/L范围内,EAHY926细胞对Tat PTD-Es的摄取有剂量依赖性,相对荧光强度从20.72AU增加到305.23AU。随着Tat PTD-Es与细胞孵育时间的延长,EAHY926对Tat PTD-Es的摄取也逐渐增加。5min-2h内荧光强度增加较为迅速,荧光强度从17.16增加到221.37,之后增加较为平缓,4h时的荧光强度增加至249.18。考察了各种胞吞途径抑制剂对Tat PTD-Es进入人脐静脉内皮细胞EAHY926的影响。细胞经4℃和NaN3(ATP抑制剂)处理后,与对照组相比,荧光信号大大减弱,荧光强度分别降低了92.49%、94.91%,说明温度、ATP在EAHY926细胞摄取Tat PTD-Es的过程中起到非常重要的作用,其入胞过程是温度、能量依赖的。而经蔗糖(网格蛋白clathrin途径抑制剂)、氯丙嗪(clathrin途径抑制剂)、木黄酮(小窝途径caveolae抑制剂)、细胞松弛素D(巨胞饮途径抑制剂)等不同摄取途径抑制剂处理后的EAHY926细胞对蛋白质的摄取均有所减弱,但是减弱的程度不尽相同。蔗糖及氯丙嗪处理后,荧光强度分别为原来的36.71%及36.86%,均减少了60%左右,提示clathrin途径也可能是Tat PTD-Es入胞机制之一。木黄酮处理后的细胞,荧光强度为原来的66.57%,降低了33%,表明Tat PTD-Es的入胞可能也与caveolae有关。细胞松弛素D处理后的细胞,荧光强度为原来的27.52%,降低了72%,提示巨胞饮也是Tat PTD-Es入胞的潜在途径。由这些结果我们推测,Tat PTD-Es入胞可能不只遵循一种途径,而是几种途径同时存在。4Tat PTD-Es穿透眼球屏障的能力与体内药效学研究小鼠分组后,分别玻璃体注射和滴眼Tat PTD-Es与Es组,生理盐水滴眼组作为空白对照。取小鼠眼球,切片,用小鼠6×His抗体作为一抗做免疫组化,检测视网膜层是否有目的蛋白。结果显示,玻璃体注射Tat PTD-Es与Es后,视网膜细胞层均有目的蛋白,表明经玻璃体注射后药物能够较快到达视网膜。滴眼给药时,Es组小鼠视网膜细胞层没有目的蛋白的分布,表明通过滴眼给药Es不能到达视网膜,而Tat PTD-Es组小鼠视网膜细胞层出现了目的蛋白,证明通过滴眼给药Tat PTD能够携带Es穿透眼球屏障到达视网膜,达到了我们的预期目的。通过激光烧伤法建立小鼠脉络膜新生血管(CNV)模型,滴眼或玻璃体注射Tat PTD-Es及Es,生理盐水滴眼组作为阴性对照,玻璃体注射Avastin作为阳性对照。实验结果表明,玻璃体注射Tat PTD-Es、Es组的小鼠眼部的CNV血管生成面积分别为(961.2±86.9)μm2和(944.6±88.3)μm2,与阴性药物组(2623.6±240.9)μm2相比CNV面积明显减少(p<0.01),且与阳性药物Avastin组(863.5±50.1)μm2相比没有显著性差异。Es滴眼给药时,CNV面积为(2514.7±260.7)μm2,与生理盐水组相比没有显著性差异,表明Es滴眼后Es不能到达眼底,不能抑制CNV的生成。而Tat PTD-Es滴眼给药组的CNV面积(1378.4±154.3)μm2,与阴性药物组相比CNV面积明显减少(p<0.01)。滴眼给药Tat PTD-Es能够抑制CNV血管的生成,此结果进一步证明了通过局部滴眼给药后Tat PTD-Es能够穿透眼球屏障到达视网膜脉络膜发挥其作用。本研究取得的成果和结论：(1)首次构建了Tat PTD-Es的毕赤酵母工程表达菌株。(2)首次构建了表达Tat PTD-Es的大肠杆菌表达系统,对蛋白质成功进行了表达,并对其包涵体进行了复性、纯化,得到了高纯度的目的蛋白。(3)复性纯化的Tat PTD-Es及Es能够明显抑制EAHY926细胞的增殖、CAM血管生成,表明其具有较好的体外活性。(4) Tat PTD-Es比Es具有更好的入胞能力,且其进入EAHY926细胞时遵循多种入胞机制。(5) Tat PTD-Es局部滴眼给药后能够穿透眼球屏障到达眼底视网膜脉络膜组织,且在体内表现出了较好的抑制脉络膜血管生成的活性。更多还原

【Abstract】 Many diseases (such as diabetic retinopathy and age-related macular degeneration) can induce vascular proliferation of eyes which makes serious damage to visual function or even results in blindness. Until now, intraocular injection or laser photocoagulation is commonly adopted to treat retinal angiogenesis in clinic. But these methods often result in serious irreversible damage on retina. Therefore, it has major social and economic significance to search some novel drugs which can prevent angiogenesis on eyes. Endostatin (Es) is an endogenous potent inhibitor of vascular endothelial cell proliferation. Its analogue, Endostar, has been approved by the State Food and Drug Administration (SFDA) in China for the treatment of patients with non-small-cell lung cancer. In addition, researchers have made some achievements of Es on prevention and treatment of ocular neovascular diseases. For example, researchers have identified that Es and Es gene can inhibit the formation of ocular neovascularization by bulbar conjunctiva injection or intravitreal injection. However, due to the presence of eye barrier, Es has to be administrated only by intraocular injection. This mode of operation is difficult and might damage the eyeball organizations, even can result in irreversible damage to patients. So it is important to look for a simple, safe, effective route of administration to treat these ocular diseases. Protein transduction domain of the transacting activator of transcription (Tat PTD) can transverse the cell membrane with different cargoes and it has the potential to cross eye barriers. Thus, in this study, Tat PTD and Es was fused together by biotechnology. And this fusion protein (Tat PTD-Es) was expected to cross ocular barriers via the mediation of Tat PTD and perform its inhibition effect on vascular proliferation of retina. The research contents and the main results are as follows.1Fusion expression and purification of Tat PTD-endostatin (Tat PTD-Es) in Pichia pastorisTat PTD-Es gene was prepared and recombinant plasmid pGAPAaA/Tat PTD-Es (pG/TE) and pGAPZaA/Es (pG/E) was constructed. Plamids was transformed into Pichia pastoris by electroporation and positive colonies were screened. The expressed product was analyzed by SDS-PAGE. The target protein was preliminarily purified with nickel affinity chromatography column.The results showed the Pichia pastoris system was constructed successfully but expressed protein was low.2Fusion expression, renaturation, purification of Tat PTD-Es in E.coliTat PTD-Es and Es were expressed in E.coli expression system. Corresponding expression vectors pET28a/Tat PTD-Es(pET28a/TE) and pET28a/Es(pET28a/E) were constructed. E. coli Rosetta (DE3) cells containing recombinant plasmid were grown at37℃. After4h induction, the cells was harvested and analyzed by SDS-PAGE. The inclusion body was renatured by direct dilution method. Subsequently, His-trapTM HP column was used to purify the protein. After purification, high purity of the target protein was obtained. The molecular weight of Tat PTD-Es was22kDa by SDS-PAGE, which was consistent with the deduced size from its coding sequence. Western blot analysis showed that Tat PTD-Es and Es reacted with6×His antibody, which showed that the fused protein has6×His tag.3Study on the activity in vitro and in vivo and endocytosis mechanism of Tat PTD-EsCCK-8assay was performed to assay the anti-proliferative effects of Tat PTD-Es and Es on endothelial cell EAHY926. Both of the two proteins significantly inhibited EAHY926proliferation in a dose dependent manner. The activity of Tat PTD-Es was higher than Es at low concentrations. Both Tat PTD-Es and Es showed more than80%inhibition on EAHY926proliferation at high concentrations(>4μmol/L).The activity of Tat PTD-Es and Es was also tested by a chick embryo chorioallantoic membrane (CAM) assay. For the control groups, the CAM were treated with saline (blank control) and bFGF (negative control) respectively. The inhibition of Tat PTD-Es and Es on angiogenesis of CAM induced by bFGF was observed. Compared with bFGF group(30.74±6.58), the number of blood vessels of CAM treated with Tat PTD-Es and Es were reduced from to (12.18±4.92) and (14.56±6.21) respectively(p<0.05). The results showed that Tat PTD-Es and Es could significantly inhibit angiogenesis of CAM induced by bFGF.The abilities of cellular internalization of Tat PTD-Es and Es were compared by fluorescence microscope and flow cytometry, respectively. Intracellular accumulation of FITC labeled proteins which incubated with EAHY926cells for4h was observed and the fluorescence intensity in cells exposed to Tat PTD-Es increased notably compared with Es. The percentage of positive cells after exposure to Tat PTD-Es and Es were99.28%and33.61%respectively. The results showed that Tat PTD significantly (p<0.05) enhanced Es delivery into EAHY926cells. The impacts of Tat PTD-Es concentration and incubation time on its uptake were studied. The uptake of Tat PTD-Es by EAHY926cells was clearly dependent on the tested concentration range of1μmol/L to10μmol/L (fluorescence intensity from20.72to305.23). In terms of the effect of the treatment time on the internalization process of Tat PTD-Es, with the incubation time increase, the fluorescence intensity inside EAHY926cells increased respectively. Rapid entrance into cells of Tat PTD-Es was observed, and the fluorescence intensity increased sharply from0min-2h (fluorescence intensity from17.16to221.37). Then it increased to249.18in a more gentle way until4h.To understand the concrete uptake pathway of Tat PTD-Es, the effects of some inhibitors on the transduction of Tat PTD-Es were investigated. Compared with the control group, the fluorescence intensity of cells that were treated with4℃and NaN3(ATP inhibitor) was greatly reduced and it decreased by92.49%and94.91%. The result indicated that ATP played an important role in the uptake pathway of Tat PTD-Es in EAHY926cells and Tat PTD-Es appeared to be taken up inside the cells via an energy-dependent active process. The uptake of Tat PTD-Es by EAHY926cells which were treated with sucrose (the inhibitor of clathrin pathway), chlorpromazine (the inhibitor of clathrin pathway), genistein (the inhibitor of caveolin pathway), cytochalasin D (the inhibitor of macropinocytosis) respectively were all decreased, but the decreases were different. When treated with sucrose and chlorpromazine, uptakes of Tat PTD-Es by EAHY926cells were all reduced by about60%. The result suggested that Tat PTD-Es entered EAHY926cell by the clathrin-mediated endocytosis. When cells were pre-incubated with genistein, the cell uptake of Tat PTD-Es was inhibited significantly by33%, indicating that a portion of Tat PTD-Es was internalized into the cells through caveolae-mediated endocytosis. The cell uptake of Tat PTD-Es was significantly reduced by72%after treating cells with cytochalasin D. The result implied that macropinocytosis might be a major uptake mechanism for Tat PTD-Es by EAHY926cells.By these results, we speculated that the cellular entrance of Tat PTD-Es into EAHY926cells may not only follow a single way, but several pathways.4Study on the ability to penetrate eye barriers and pharmacodynamics in vivo of Tat PTD-EsAfter mice were grouped, Tat PTD-Es or Es was eye-dropped or intravitreally injected. Physiological saline was eye-dropped as control. Mouse eyes were harvested and the fusion protein was examined with6×His antibody by immunohistochemistry. The results showed that both Tat PTD-Es and Es appeared in retina after intravitreal injection. It indicated that drugs can quickly reach the retinal after intravitreal injection. But Es did not appear in retina after eye-dropping. It was implicated that Es can not arrive in retina through eye-dropping. Tat PTD-Es appeared in retinal after eye-dropping. It was verified Tat PTD can carry protein to penetrate eye barriers through eye-dropping. These results achieved the intend purpose.Mouse choroidal neovascularization (CNV) models were established by laser-burning. Then Tat PTD-Es or Es was eye-dropped or intravitreally injected. Physiological saline was eye-dropped as negative control, and Avastin was intravitreally injected as positive control. The results indicated that the CNV areas of mouse intravitreally injected with Tat PTD-Es and Es were respectively (961.2±86.9)μm2and (944.6±88.3)μm2, which obviously decreased comparing to negative control, of which the CNV area was(2623.6±240.9)μm2(p<0.01), and was not significantly different from positive control with an CNV area of (863.5±50.1)μm2. When Es was eye-dropped, the CNV area was (2514.7±260.)μm2. It was not significantly different from the negative control, showing no inhibition of CNV angiogenesis. The CNV area of Tat PTD-Es eye-dropping mice was (1378.4±154.3)μm2, which significantly decreased compared to negative control (p<0.01). It had significant differences from those of intravitreal injection of positive control Avastin and Tat PTD-Es. Tat PTD-Es via eye drops was able to inhibit CNV angiogenesis, and these results further demonstrated that Tat PTD-Es had the capacity to penetrate the eye barrier, reach the retina choroid and play its role via eye-dropping.Results and conclusions:(1) The expression strain of Tat PTD Es in Pichia was constructed.(2) The expression strain of Tat PTD-Es in E.coli was firstly constructed. The protein was expressed successfully and high purity of target proteins was obtained by renaturation and purification from inclusion bodies.(3) Tat PTD-Es and Es had excellent activity in vitro and they can significantly inhibit EAHY926endothelial cell proliferation and CAM angiogenesis.(4) Tat PTD-Es has better cell penetrating ability than Es and it followed several cellular mechanisms entering into EAHY926.(5) Tat PTD-Es could penetrate the eye barrier and reached the retina choroid via eye drops. It also showed a better inhibition choroidal angiogenesis in vivo.更多还原