促肝细胞生长素靶向脂质体的制备及其抗肝纤维化作用的研究
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摘要
肝纤维化以及肝硬化代表着各种慢性肝病的最终共同转归。肝星状细胞(hepatic stellate cells,HSC)是肝纤维化过程中肝脏内合成大量胶原纤维的首要细胞,因此是抗纤维化治疗的靶细胞。增加药物对于HSC的靶向性可以增加药物的抗纤维化效果。
含有精氨酸—甘氨酸—天门冬氨酸(Arg-Gly-Asp,RGD)系列的环肽可以特异地识别位于活化HSC表面的Ⅵ型胶原受体,因此可以用来修饰作用于活化HSC的药物。立体稳定脂质体(sterically stabilized liposomes,SSL)由于表面被聚乙二醇修饰从而避免被网状内皮系统识别和清除,因此可以延长脂质体内包封药物的作用时间。促肝细胞生长素(promoting hepatocyte growth factor,pHGF)属于促肝细胞生长因子类物质(该类物质还包括重组的肝细胞生长因子),体内外实验发现具有抗肝纤维化的作用;但是由于半衰期短,并且可作用于其它细胞引起不利作用如促进肝肿瘤生长,因此在临床应用中未表现出满意的抗纤维化治疗效果。
本研究的主要目的是制备RGD环肽修饰的包封pHGF的SSL(RGD-SSL-pHGF),拟通过SSL的包封延长pHGF的作用时间,并通过RGD环肽的修饰增加pHGF对活化HSC的靶向性。通过体内外实验评价RGD-SSL-pHGF的抗纤维化作用及可能的作用机制。
本研究的主要内容包括:促肝细胞生长素和重组肝细胞生长因子对肝星状细胞作用的比较;RGD环肽修饰的包封pHGF的立体稳定脂质体的制备及其理化性质的检测;RGD-SSL-pHGF对活化肝星状细胞的作用;RGD-SSL-pHGF对大鼠肝纤维化的治疗作用。
第一部分促肝细胞生长素和重组肝细胞生长因子对肝星状细胞作用的比较
目的:比较国产的促肝细胞生长素(商品名威佳)和国外进口的重组人肝细胞生长因子(recombinant human hepatocyte growth factor,rh-HGF)对活化的HSC生长和凋亡的作用。
方法:采用链酶蛋白酶E/Ⅳ型胶原酶灌注——密度梯度离心的方法从正常雄性SD大鼠(400～450g,14周龄)肝脏分离HSC,培养传代后检测α—平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)表达的情况。四甲基偶氮唑蓝(3-(4,5-dimethylthiazo(-z-y1)-3,5 diphenytetrazoliumromide,MTT)法检测pHGF和rh-HGF对HSC生长的影响,TUNEL(TdT-mediated dUTP Nick-EndLabeling)法检测这两种生长因子对活化的HSC凋亡诱导的情况。
结果:从正常大鼠肝脏分离HSC,平均每只大鼠分得的细胞数为2×10~7,细胞活力为96%。通过培养时形态的观察和免疫组化检测α-SMA的表达,提示传代培养24小时的细胞为活化的HSC。pHGF和rh-HGF都能抑制HSC的生长并且随着药物浓度的增加,抑制作用逐渐增强;相同药物浓度时,rh-HGF的抑制作用强于pHGF;pHGF的IC_(50)为165.67±7.09ng/ml,高于rh-HGF(132.00±4.20ng/ml)(P=0.02)。150ng/ml的pHGF和rh-HGF都诱导活化的HSC发生凋亡,HSC的凋亡率为分别为33.67%±1.34%和43.96%±2.79%(P=0.05)。
结论:pHGF和rh-HGF都抑制了HSC的生长和促进活化HSC凋亡,rh-HGF的作用强于pHGF。
第二部分RGD环肽修饰的包封pHGF的立体稳定脂质体的制备及其理化性质的检测
目的:制备包封pHGF的RGD环肽修饰的立体稳定脂质体(stericallystabilized lliposomes,SSL)(RGD-SSL-pHGF),评价其理化性质。
方法:采用薄膜法结合膜挤压法制备包封pHGF的SSL,RGD环肽通过与位于脂质体表面的二油酰磷脂酰乙醇胺—聚乙二醇—马来酰亚胺(DOPE-PEG-MAL)结合后修饰脂质体,从而制备RGD-SSL-pHGF,评价该脂质体的粒径、包封率和稳定性等重要指标。此外,制备包封钙黄绿素(calcein,CF)的无RGD环肽修饰的SSL(SSL-CF)和有RGD环肽修饰的SSL(RGD-SSL-CF),含有SSL-CF、RGD-SSL-CF和单纯CF溶液的培养液培养活化HSC24小时后,流式细胞仪检测HSC内荧光强度从而评价RGD环肽的靶向性。
结果:制得的RGD-SSL-pHGF粒径为92.7nm,表面电位为—18.49mv;包封率为32.38%±0.09%,即每ml脂质体溶液约含有pHGF 5μg,RGD环肽10nmol;在4℃以下条件保存,该脂质体稳定;制备过程中磷脂损失6.58%。RGD-SSL-CF、SSL-CF和CF与HSC培养24小时后,RGD-SSL-CF组的HSC内荧光强度显著高于SSL-CF组和CF组(P＜0.05)。
结论:成功制备了粒径均一的RGD-SSL-CF,RGD修饰的SSL延长了所包封药物的作用时间并具有HSC靶向性。
第三部分RGD-SSL-pHGF对活化肝星状细胞的作用
目的:利用体外培养的HSC比较RGD-SSL-pHGF、无RGD环肽修饰的包封pHGF的SSL(SSL-pHGF)、pHGF和不包封pHGF的RGD环肽修饰的SSL(RGD-SSL)对活化的HSC的作用。
方法:照第二部分的方法制备RGD-SSL-pHGF、SSL-pHGF和RGD-SSL。MTT法比较三种脂质体和pHGF对HSC生长的抑制情况,TUNEL法比较对活化的HSC凋亡诱导情况,荧光定量RT-PCR比较对HSC内α-SMA、α1(Ⅰ)型前胶原(procollagenα1(Ⅰ))、组织金属蛋白酶抑制物—1(tissue inhibitor ofmetalloproteinase-1,TIMP-1)、TIMP-2、基质金属蛋白酶-2(matrixmetalloproteinase-2,MMP-2)、转化生长因子—β1(transforming growthfactor-β1,TGF-β1)的mRNA表达的抑制情况,Western blot比较对α-SMA和Ⅰ型胶原生成的抑制情况。
结果:pHGF在150ng/ml浓度条件下,与大鼠HSC培养48小时后,促进HSC的凋亡并抑制HSC内α-SMA、TGF-β1、α_1(Ⅰ)型前胶原、TIMP-1、TIMP-2和MMP-2的mRNA表达,同时抑制了α-SMA和Ⅰ型胶原的生成。与SSL-pHGF、pHGF和RGD-SSL相比,RGD-SSL-pHGF更显著地抑制HSC的生长,诱导更多活化HSC发生凋亡,同时更显著地抑制了HSC内相关检测基因的表达以及α-SMA和Ⅰ型胶原的生成。
结论:RGD环肽的修饰和SSL的包封增强了pHGF对活化HSC的抗纤维化作用。
第四部分RGD-SSL-pHGF对大鼠肝纤维化的治疗作用
目的:评价RGD-SSL-pHGF对大鼠肝纤维化的治疗作用及作用机制。
方法:腹腔注射硫代乙酰胺10周诱导大鼠出现肝纤维化。休息3周后,60只肝纤维化大鼠分成六组(n=10):RGD-SSL-pHGF组、SSL-pHGF组、pHGF组、RGD-SSL组、pHGF+RGD-SSL组和注射生理盐水的模型对照组。每隔一天注射一次相应的药物,共4周。比较每组的肝脏病理分期、胶原纤维面积百分比、每克肝组织羟脯氨酸的含量、α-SMA阳性细胞凋亡率和α_1(Ⅰ)型前胶原、α_1(Ⅲ)型前胶原mRNA的表达情况。
结果:与其他组相比,注射RGD-SSL-pHGF的大鼠肝纤维化明显缓解,胶原纤维面积百分比显著下降,每克肝组织的羟脯氨酸含量明显减少。RGD-SSL-pHGF诱导更多的α-SMA阳性细胞发生凋亡,更明显抑制了α_1(Ⅰ)型前胶原mRNA的表达。
结论:RGD-SSL-pHGF通过诱导α-SMA阳性细胞凋亡、促进胶原纤维的降解和抑制Ⅰ型胶原的生成从而促进肝纤维化的逆转。
Liver fibrosis and advanced fibrosis(liver cirrhosis) represent the final common pathway of virtually all chronic liver diseases.The hepatic stellate cell(HSC) is the primary cell-type in the liver responsible for excess collagen synthesis during hepatic fibrosis,so this cell is the major target for anti-fibrosis treatment.The anti-fibrosis effect will be enhanced by enhancing drugs target to HSC.
Cyclic RGD(Arg-Gly-Asp) peptides recognize specially the typeⅥcollagen receptor which is on the surface of activated HSC,so they can be used to modify the drugs which have effects on activated HSC.After modified with polyethylene glycol (PEG),sterically stabilized liposomes(SSL) have the ability to escape the recognization and clearance of reticuloendothelial system so that they can prolong the half-life phase of drug which is encapsulated.Studies in vitro and in vivo have shown that promoting hepatocyte growth factor(pHGF) has the anti-fibrosis effect on liver fibrosis,but pHGF has not shown a satisfactory anti-fibrosis effect in clinical application because of its short half-life phase and its adverse events caused by affecting other cells,for example promoting the development of liver tumor.
Therefore,the purpose of our study is to synthesis a kind of SSL which is encapsulating pHGF and modified with cyclic RGD peptides(RGD-SSL-pHGF). Thus the half-life period of pHGF will be prolonged and its target to activated HSC will be enhanced.The anti-fibrosis effect of RGD-SSL-pHGF and the mechanism of action will be evaluated.
This study includes four parts:to compare the effect of pHGF and recombinant hepatocyte growth factor on HSC;to synthesis RGD-SSL-pHGF and evaluate their physico-chemical properties;to evaluate the effect of RGD-SSL-pHGF on culture-activated HSC;to evaluate the treatment effect of RGD-SSL-pHGF on liver fibrosis in rats.
Part One Compare the effect of pHGF and recombinant hepatocyte growth factor on HSC.
Aims:To compare the effect of pHGF and recombinant human hepatocyte growth factor(rh-HGF) on the growth and apoptosis of culture-activated HSC.
Methods:HSC were isolated from normal male SD rats(400～450g,14 weeks old) by continuous infusion with pronase E and typeⅣcollagenase and purified with density gradient centrifugation.After cultured and passaged,the expression ofα-smooth muscle actin(α-SMA) in HSC was evaluated.Then the growth of HSC inhibited by pHGF and rh-HGF were compared with 3-(4,5-dimethylthiazo(-z-yl)-3,5 diphenytetrazoliumromide(MTT) method and the apoptosis of activated HSC promoted by pHGF and rh-HGF were compared with TdT-mediated dUTP Nick-End Labeling(TUNEL) method.
Results:The harvest rate of HSC was about 2×10~7 per rat,and the viability was about 96%.The cells cultured for 24 hours after passaged were proved to be activated HSC based on the appearance and the expression ofα-SMA.Both pHGF and rh-HGF inhibited the growth of HSC,and this effect was increasing following with the increased concentration.The inhibition effect of rh-HGF was superior to that of pHGF at the same concentration.The IC_(50) of pHGF was 165.67±7.09ng/ml,higher than that of rh-HGF(132.00±4.20ng/ml)(P=0.02).pHGF induced 33.67%activated HSC to apoptosis,while rh-HGF induced 43.96%activated HSC to apoptosis at the concentration of 150ng/ml(P=0.05).
Conclusion:Both pHGF and rh-HGF inhibited the growth of HSC and promoted the apoptosis of activated HSC,but rh-HGF was superior to pHGF.
Part Two Synthesis RGD-SSL-pHGF and evaluate their physico-chemical properties.
Aims:To synthesis RGD-SSL-pHGF and evaluate their physico-chemical properties.
Methods:SSL encapsulating pHGF were synthesized,and modified with cyclic RGD peptides which were conjugated with DOPE-PEG-MAL on the surface of SSL. Then the properties of RGD-SSL-pHGF were evaluated,including size,encapsulated percentage of pHGF and stability.In addition,SSL encapsulating calcein(CF) modified with or without RGD(RGD-SSL-CF or SSL-CF) were synthesized.After cultured with these liposomes and CF solution for 24 hours,the fluorescence intensity in HSC cytoplasm was evaluated with flow cytometry in order to evaluate the target of these agents to HSC.
Results:The mean size of RGD-SSL-pHGF was 92.7nm and their surface potential was—18.49mv.The encapsulated percentage of pHGF was 32.38%,that was to say,there were about 5μg pHGF and 10nmol RGD per milliliter liposomes solution.These liposomes were stable at below 4℃.6.58%phospholipids were loss in the synthesize process.After cultured with RGD-SSL-CF,SSL-CF and CF solution for 24 hours,the fluorescence intensity in RGD-SSL-CF group was highest(P＜0.05).
Conclusion:RGD-SSL-pHGF were synthesized.The encapsulation in SSL modified with RGD prolonged the action time of pHGF and enhanced its target to activated HSC.
Part Three The effect of RGD-SSL-pHGF on culture-activated HSC
Aims:To compare the effects of pHGF encapsulated in SSL modified with or without RGD(RGD-SSL-pHGF or SSL-pHGF),pHGF and SSL modified with RGD (RGD-SSL) on cultured-activated HSC.
Methods:RGD-SSL-pHGF,SSL-pHGF and RGD-SSL were synthesized.After cultured with these liposomes and pHGF,the growth of HSC,the expression of mRNA and the production ofα-SMA and typeⅠcollagen in HSC were compared.The apoptosis of HSC was compared,too.
Results:After culturing with HSC for 48 hours,pHGF promoted the apoptosis of HSC and inhibited the expression ofα-SMA,transforming growth factor-β1, procollagenα_1(Ⅰ),tissue inhibitor of metalloproteinase-1 and -2,and matrix metalloproteinase-2 mRNA.It also inhibited the production ofα-SMA and typeⅠcollagen.Compared with SSL-pHGF,pHGF and RGD-SSL,RGD-SSL-pHGF has the most significant effects on HSC.
Conclusion:After encapsulated into SSL modified with RGD,pHGF showed more significant anti-fibrosis effect on activated HSC.
Part Four The treatment effect of RGD-SSL-pHGF on liver fibrosis in rats
Aims:To evaluated the treatment effect and the action mechanism of RGD-SSL-pHGF on liver fibrosis in rats.
Methods:Liver fibrosis was induced in rats by injecting thioacetamide intraperitoneally for 10 weeks.After resting for another 3 weeks,these rats were divided into 6 groups(n=10) according to injected agents:RGD-SSL-pHGF, SSL-pHGF,pHGF,RGD-SSL,pHGF plus RGD-SSL and saline.After injected these agents every other days for 4 weeks,the histologic stage,the fibrotic area percentage, the hydroxyproline amount per gram hepatic tissue,the apoptotic rate ofα-SMA-positive cells and the expression of procollagenα_1(Ⅰ) andα_1(Ⅲ) mRNA in every group were compared.
Results:The resolution of liver fibrosis was the most significant in RGD-SSL-pHGF group.There were the lowest fibrotic area percentage and the least hydroxyproline amount in RGD-SSL-pHGF group,too.The injection of RGD-SSL-pHGF also promoted the mostα-SMA-positive cells to apoptosis and inhibited most significantly the expression of procollagenα_1(Ⅰ) mRNA.
Conclusion:RGD-SSL-pHGF promoted the resolution of liver fibrosis by promoting the apoptosis ofα-SMA-positive cells and the digestion of existing fiber collagen and inhibiting the production of typeⅠcollagen.
含有精氨酸—甘氨酸—天门冬氨酸(Arg-Gly-Asp,RGD)系列的环肽可以特异地识别位于活化HSC表面的Ⅵ型胶原受体,因此可以用来修饰作用于活化HSC的药物。立体稳定脂质体(sterically stabilized liposomes,SSL)由于表面被聚乙二醇修饰从而避免被网状内皮系统识别和清除,因此可以延长脂质体内包封药物的作用时间。促肝细胞生长素(promoting hepatocyte growth factor,pHGF)属于促肝细胞生长因子类物质(该类物质还包括重组的肝细胞生长因子),体内外实验发现具有抗肝纤维化的作用;但是由于半衰期短,并且可作用于其它细胞引起不利作用如促进肝肿瘤生长,因此在临床应用中未表现出满意的抗纤维化治疗效果。
本研究的主要目的是制备RGD环肽修饰的包封pHGF的SSL(RGD-SSL-pHGF),拟通过SSL的包封延长pHGF的作用时间,并通过RGD环肽的修饰增加pHGF对活化HSC的靶向性。通过体内外实验评价RGD-SSL-pHGF的抗纤维化作用及可能的作用机制。
本研究的主要内容包括:促肝细胞生长素和重组肝细胞生长因子对肝星状细胞作用的比较;RGD环肽修饰的包封pHGF的立体稳定脂质体的制备及其理化性质的检测;RGD-SSL-pHGF对活化肝星状细胞的作用;RGD-SSL-pHGF对大鼠肝纤维化的治疗作用。
第一部分促肝细胞生长素和重组肝细胞生长因子对肝星状细胞作用的比较
目的:比较国产的促肝细胞生长素(商品名威佳)和国外进口的重组人肝细胞生长因子(recombinant human hepatocyte growth factor,rh-HGF)对活化的HSC生长和凋亡的作用。
方法:采用链酶蛋白酶E/Ⅳ型胶原酶灌注——密度梯度离心的方法从正常雄性SD大鼠(400～450g,14周龄)肝脏分离HSC,培养传代后检测α—平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)表达的情况。四甲基偶氮唑蓝(3-(4,5-dimethylthiazo(-z-y1)-3,5 diphenytetrazoliumromide,MTT)法检测pHGF和rh-HGF对HSC生长的影响,TUNEL(TdT-mediated dUTP Nick-EndLabeling)法检测这两种生长因子对活化的HSC凋亡诱导的情况。
结果:从正常大鼠肝脏分离HSC,平均每只大鼠分得的细胞数为2×10~7,细胞活力为96%。通过培养时形态的观察和免疫组化检测α-SMA的表达,提示传代培养24小时的细胞为活化的HSC。pHGF和rh-HGF都能抑制HSC的生长并且随着药物浓度的增加,抑制作用逐渐增强;相同药物浓度时,rh-HGF的抑制作用强于pHGF;pHGF的IC_(50)为165.67±7.09ng/ml,高于rh-HGF(132.00±4.20ng/ml)(P=0.02)。150ng/ml的pHGF和rh-HGF都诱导活化的HSC发生凋亡,HSC的凋亡率为分别为33.67%±1.34%和43.96%±2.79%(P=0.05)。
结论:pHGF和rh-HGF都抑制了HSC的生长和促进活化HSC凋亡,rh-HGF的作用强于pHGF。
第二部分RGD环肽修饰的包封pHGF的立体稳定脂质体的制备及其理化性质的检测
目的:制备包封pHGF的RGD环肽修饰的立体稳定脂质体(stericallystabilized lliposomes,SSL)(RGD-SSL-pHGF),评价其理化性质。
方法:采用薄膜法结合膜挤压法制备包封pHGF的SSL,RGD环肽通过与位于脂质体表面的二油酰磷脂酰乙醇胺—聚乙二醇—马来酰亚胺(DOPE-PEG-MAL)结合后修饰脂质体,从而制备RGD-SSL-pHGF,评价该脂质体的粒径、包封率和稳定性等重要指标。此外,制备包封钙黄绿素(calcein,CF)的无RGD环肽修饰的SSL(SSL-CF)和有RGD环肽修饰的SSL(RGD-SSL-CF),含有SSL-CF、RGD-SSL-CF和单纯CF溶液的培养液培养活化HSC24小时后,流式细胞仪检测HSC内荧光强度从而评价RGD环肽的靶向性。
结果:制得的RGD-SSL-pHGF粒径为92.7nm,表面电位为—18.49mv;包封率为32.38%±0.09%,即每ml脂质体溶液约含有pHGF 5μg,RGD环肽10nmol;在4℃以下条件保存,该脂质体稳定;制备过程中磷脂损失6.58%。RGD-SSL-CF、SSL-CF和CF与HSC培养24小时后,RGD-SSL-CF组的HSC内荧光强度显著高于SSL-CF组和CF组(P＜0.05)。
结论:成功制备了粒径均一的RGD-SSL-CF,RGD修饰的SSL延长了所包封药物的作用时间并具有HSC靶向性。
第三部分RGD-SSL-pHGF对活化肝星状细胞的作用
目的:利用体外培养的HSC比较RGD-SSL-pHGF、无RGD环肽修饰的包封pHGF的SSL(SSL-pHGF)、pHGF和不包封pHGF的RGD环肽修饰的SSL(RGD-SSL)对活化的HSC的作用。
方法:照第二部分的方法制备RGD-SSL-pHGF、SSL-pHGF和RGD-SSL。MTT法比较三种脂质体和pHGF对HSC生长的抑制情况,TUNEL法比较对活化的HSC凋亡诱导情况,荧光定量RT-PCR比较对HSC内α-SMA、α1(Ⅰ)型前胶原(procollagenα1(Ⅰ))、组织金属蛋白酶抑制物—1(tissue inhibitor ofmetalloproteinase-1,TIMP-1)、TIMP-2、基质金属蛋白酶-2(matrixmetalloproteinase-2,MMP-2)、转化生长因子—β1(transforming growthfactor-β1,TGF-β1)的mRNA表达的抑制情况,Western blot比较对α-SMA和Ⅰ型胶原生成的抑制情况。
结果:pHGF在150ng/ml浓度条件下,与大鼠HSC培养48小时后,促进HSC的凋亡并抑制HSC内α-SMA、TGF-β1、α_1(Ⅰ)型前胶原、TIMP-1、TIMP-2和MMP-2的mRNA表达,同时抑制了α-SMA和Ⅰ型胶原的生成。与SSL-pHGF、pHGF和RGD-SSL相比,RGD-SSL-pHGF更显著地抑制HSC的生长,诱导更多活化HSC发生凋亡,同时更显著地抑制了HSC内相关检测基因的表达以及α-SMA和Ⅰ型胶原的生成。
结论:RGD环肽的修饰和SSL的包封增强了pHGF对活化HSC的抗纤维化作用。
第四部分RGD-SSL-pHGF对大鼠肝纤维化的治疗作用
目的:评价RGD-SSL-pHGF对大鼠肝纤维化的治疗作用及作用机制。
方法:腹腔注射硫代乙酰胺10周诱导大鼠出现肝纤维化。休息3周后,60只肝纤维化大鼠分成六组(n=10):RGD-SSL-pHGF组、SSL-pHGF组、pHGF组、RGD-SSL组、pHGF+RGD-SSL组和注射生理盐水的模型对照组。每隔一天注射一次相应的药物,共4周。比较每组的肝脏病理分期、胶原纤维面积百分比、每克肝组织羟脯氨酸的含量、α-SMA阳性细胞凋亡率和α_1(Ⅰ)型前胶原、α_1(Ⅲ)型前胶原mRNA的表达情况。
结果:与其他组相比,注射RGD-SSL-pHGF的大鼠肝纤维化明显缓解,胶原纤维面积百分比显著下降,每克肝组织的羟脯氨酸含量明显减少。RGD-SSL-pHGF诱导更多的α-SMA阳性细胞发生凋亡,更明显抑制了α_1(Ⅰ)型前胶原mRNA的表达。
结论:RGD-SSL-pHGF通过诱导α-SMA阳性细胞凋亡、促进胶原纤维的降解和抑制Ⅰ型胶原的生成从而促进肝纤维化的逆转。
Liver fibrosis and advanced fibrosis(liver cirrhosis) represent the final common pathway of virtually all chronic liver diseases.The hepatic stellate cell(HSC) is the primary cell-type in the liver responsible for excess collagen synthesis during hepatic fibrosis,so this cell is the major target for anti-fibrosis treatment.The anti-fibrosis effect will be enhanced by enhancing drugs target to HSC.
Cyclic RGD(Arg-Gly-Asp) peptides recognize specially the typeⅥcollagen receptor which is on the surface of activated HSC,so they can be used to modify the drugs which have effects on activated HSC.After modified with polyethylene glycol (PEG),sterically stabilized liposomes(SSL) have the ability to escape the recognization and clearance of reticuloendothelial system so that they can prolong the half-life phase of drug which is encapsulated.Studies in vitro and in vivo have shown that promoting hepatocyte growth factor(pHGF) has the anti-fibrosis effect on liver fibrosis,but pHGF has not shown a satisfactory anti-fibrosis effect in clinical application because of its short half-life phase and its adverse events caused by affecting other cells,for example promoting the development of liver tumor.
Therefore,the purpose of our study is to synthesis a kind of SSL which is encapsulating pHGF and modified with cyclic RGD peptides(RGD-SSL-pHGF). Thus the half-life period of pHGF will be prolonged and its target to activated HSC will be enhanced.The anti-fibrosis effect of RGD-SSL-pHGF and the mechanism of action will be evaluated.
This study includes four parts:to compare the effect of pHGF and recombinant hepatocyte growth factor on HSC;to synthesis RGD-SSL-pHGF and evaluate their physico-chemical properties;to evaluate the effect of RGD-SSL-pHGF on culture-activated HSC;to evaluate the treatment effect of RGD-SSL-pHGF on liver fibrosis in rats.
Part One Compare the effect of pHGF and recombinant hepatocyte growth factor on HSC.
Aims:To compare the effect of pHGF and recombinant human hepatocyte growth factor(rh-HGF) on the growth and apoptosis of culture-activated HSC.
Methods:HSC were isolated from normal male SD rats(400～450g,14 weeks old) by continuous infusion with pronase E and typeⅣcollagenase and purified with density gradient centrifugation.After cultured and passaged,the expression ofα-smooth muscle actin(α-SMA) in HSC was evaluated.Then the growth of HSC inhibited by pHGF and rh-HGF were compared with 3-(4,5-dimethylthiazo(-z-yl)-3,5 diphenytetrazoliumromide(MTT) method and the apoptosis of activated HSC promoted by pHGF and rh-HGF were compared with TdT-mediated dUTP Nick-End Labeling(TUNEL) method.
Results:The harvest rate of HSC was about 2×10~7 per rat,and the viability was about 96%.The cells cultured for 24 hours after passaged were proved to be activated HSC based on the appearance and the expression ofα-SMA.Both pHGF and rh-HGF inhibited the growth of HSC,and this effect was increasing following with the increased concentration.The inhibition effect of rh-HGF was superior to that of pHGF at the same concentration.The IC_(50) of pHGF was 165.67±7.09ng/ml,higher than that of rh-HGF(132.00±4.20ng/ml)(P=0.02).pHGF induced 33.67%activated HSC to apoptosis,while rh-HGF induced 43.96%activated HSC to apoptosis at the concentration of 150ng/ml(P=0.05).
Conclusion:Both pHGF and rh-HGF inhibited the growth of HSC and promoted the apoptosis of activated HSC,but rh-HGF was superior to pHGF.
Part Two Synthesis RGD-SSL-pHGF and evaluate their physico-chemical properties.
Aims:To synthesis RGD-SSL-pHGF and evaluate their physico-chemical properties.
Methods:SSL encapsulating pHGF were synthesized,and modified with cyclic RGD peptides which were conjugated with DOPE-PEG-MAL on the surface of SSL. Then the properties of RGD-SSL-pHGF were evaluated,including size,encapsulated percentage of pHGF and stability.In addition,SSL encapsulating calcein(CF) modified with or without RGD(RGD-SSL-CF or SSL-CF) were synthesized.After cultured with these liposomes and CF solution for 24 hours,the fluorescence intensity in HSC cytoplasm was evaluated with flow cytometry in order to evaluate the target of these agents to HSC.
Results:The mean size of RGD-SSL-pHGF was 92.7nm and their surface potential was—18.49mv.The encapsulated percentage of pHGF was 32.38%,that was to say,there were about 5μg pHGF and 10nmol RGD per milliliter liposomes solution.These liposomes were stable at below 4℃.6.58%phospholipids were loss in the synthesize process.After cultured with RGD-SSL-CF,SSL-CF and CF solution for 24 hours,the fluorescence intensity in RGD-SSL-CF group was highest(P＜0.05).
Conclusion:RGD-SSL-pHGF were synthesized.The encapsulation in SSL modified with RGD prolonged the action time of pHGF and enhanced its target to activated HSC.
Part Three The effect of RGD-SSL-pHGF on culture-activated HSC
Aims:To compare the effects of pHGF encapsulated in SSL modified with or without RGD(RGD-SSL-pHGF or SSL-pHGF),pHGF and SSL modified with RGD (RGD-SSL) on cultured-activated HSC.
Methods:RGD-SSL-pHGF,SSL-pHGF and RGD-SSL were synthesized.After cultured with these liposomes and pHGF,the growth of HSC,the expression of mRNA and the production ofα-SMA and typeⅠcollagen in HSC were compared.The apoptosis of HSC was compared,too.
Results:After culturing with HSC for 48 hours,pHGF promoted the apoptosis of HSC and inhibited the expression ofα-SMA,transforming growth factor-β1, procollagenα_1(Ⅰ),tissue inhibitor of metalloproteinase-1 and -2,and matrix metalloproteinase-2 mRNA.It also inhibited the production ofα-SMA and typeⅠcollagen.Compared with SSL-pHGF,pHGF and RGD-SSL,RGD-SSL-pHGF has the most significant effects on HSC.
Conclusion:After encapsulated into SSL modified with RGD,pHGF showed more significant anti-fibrosis effect on activated HSC.
Part Four The treatment effect of RGD-SSL-pHGF on liver fibrosis in rats
Aims:To evaluated the treatment effect and the action mechanism of RGD-SSL-pHGF on liver fibrosis in rats.
Methods:Liver fibrosis was induced in rats by injecting thioacetamide intraperitoneally for 10 weeks.After resting for another 3 weeks,these rats were divided into 6 groups(n=10) according to injected agents:RGD-SSL-pHGF, SSL-pHGF,pHGF,RGD-SSL,pHGF plus RGD-SSL and saline.After injected these agents every other days for 4 weeks,the histologic stage,the fibrotic area percentage, the hydroxyproline amount per gram hepatic tissue,the apoptotic rate ofα-SMA-positive cells and the expression of procollagenα_1(Ⅰ) andα_1(Ⅲ) mRNA in every group were compared.
Results:The resolution of liver fibrosis was the most significant in RGD-SSL-pHGF group.There were the lowest fibrotic area percentage and the least hydroxyproline amount in RGD-SSL-pHGF group,too.The injection of RGD-SSL-pHGF also promoted the mostα-SMA-positive cells to apoptosis and inhibited most significantly the expression of procollagenα_1(Ⅰ) mRNA.
Conclusion:RGD-SSL-pHGF promoted the resolution of liver fibrosis by promoting the apoptosis ofα-SMA-positive cells and the digestion of existing fiber collagen and inhibiting the production of typeⅠcollagen.
引文
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