摘要
背景及目的
肝纤维化是慢性肝病的一种常见的病理变化，其主要特征是细胞外基质(extracellularmatrix, ECM)在肝内的积累。受到慢性肝损伤因子刺激后，肝星状细胞(hepatic stellatecell，HSC)从静止状态活化为能表达α-平滑肌肌动蛋白(alpha-smooth muscle actin,α-SMA)、产生大量ECM的肌成纤维样细胞，从而促进肝纤维化。转化生长因子(Transforming growth factor β1, TGF-β1)一直被认为是一个最有力的促进肝纤维化的细胞介质，但是由于它效应复杂，完全阻断可能会产生严重的副作用。结缔组织生长因子(connective tissue growth factor, CTGF)作为TGF-β1直接下游介质，其作用较为单一，与TGF-β1相比，其生物学效应更局限于纤维化的发生，近年来逐步受到关注。CTGF不仅可介导原代HSC的活化、增殖和迁移，还能促进活化HSC分泌ECM。研究表明抑制CTGF的表达可以明显抑制HSC的活化、增殖，减少ECM的合成。CTGF在正常肝组织表达水平很低，在纤维化肝脏，其表达水平明显增高，与肝纤维化的发生发展密切相关。通过小干扰RNA(small interfering RNA，siRNA)阻断CTGF策略取得了良好的抗肝纤维化作用。
目前，RNA干扰技术的主要效应分子为siRNA、short hairpin RNA(shRNA)和微小RNA(microRNA，miRNA)。人工microRNA(artificial microRNA，amiRNA)是指利用天然内源性miRNA的骨干生成成熟miRNA，有效沉默目的基因。实验表明amiRNA较siRNA或shRNA更安全、有效。然而，面临的挑战是如何有效地将amiRNAs传递到靶器官。
近几年大量研究报道超声靶向微泡破坏(ultrasound-targeted microbubble destruction,UTMD)能有效、安全增强基因转染效率并具有高度的靶向性，是一种新型的基因传递方法。微泡可作为基因的载体，在超声辐照作用下，微泡破坏，基因可在靶区释放，同时产生的空化效应增加细胞膜的通透性，促进基因进入细胞。但是目前普通微泡存在携带基因不足的缺陷。研究表明将纳米脂质体连接到微泡的表面，可以提高微泡的基因携带量。
因此，本研究模仿内源性miRNA构建针对CTGF的人工microRNA质粒载体，观察在体外对大鼠肝星状细胞(HSC-T6)中CTGF的沉默效果；探讨高机械指数诊断超声联合微泡对肝脏通透性的影响及介导基因转染的优化条件；采用生物素-亲和素-生物素连接的方法，将载基因生物素化阳离子脂质体与生物素化微泡耦联制备出一种新型载基因微泡载体；采用新型超声微泡载体介导靶向CTGF的人工microRNA，利用UTMD技术介导其体内转染，旨在探讨其体内抑制大鼠肝CTGF基因表达的可行性及用于抗肝纤维化的有效性。方法
1.靶向CTGF的人工microRNA质粒载体的构建和干扰筛选：
模拟内源性miRNA，设计四对靶向大鼠CTGF的miRNA前体序列，同时设计一对阴性对照序列，构建到pcDNA6.2-GW/EmGFPmiR质粒载体中，并对重组质粒进行测序。重组质粒分别命名为miRNA-CTGF-1，miRNA-CTGF-2，miRNA-CTGF-3，miRNA-CTGF-4和miRNA-CTGF-Scramble。
用脂质体转染法将以上质粒分别转染入大鼠肝星状细胞系HSC-T6细胞中，同时设未进行质粒转染的空白对照组，转染24h后倒置荧光显微镜观察转染效率，转染48h后荧光定量PCR法检测HSC-T6中CTGF mRNA表达情况，72h后Western Blot法检测CTGF蛋白表达情况，从而筛选出最高效沉默CTGF基因表达的干扰质粒载体。
2.高机械指数诊断超声联合微泡对大鼠肝脏通透性的作用及安全性研究
SD雄性大鼠104只随机分为四组：超声联合微泡组(US+MB组)、单纯超声组(US组)、单纯微泡组(MB组)、对照组(control组)。采用频率为1.5/3.2MHz，机械指数(MI)=1.0的高机械指数诊断超声进行辐照。用伊文氏蓝(Evans blue，EB)为示踪剂检测高机械指数诊断超声联合微泡对正常大鼠肝血管通透性的影响，激光共聚焦显微镜直观观察不同处理组EB在大鼠肝组织内的分布，硝酸镧示踪法透射电镜观察肝细胞膜通透性的变化。检测处理前及处理后0.5h、12h、24h血中丙氨酸氨基转移酶(alanine aminotransferase，ALT)和天冬氨酸氨基转移酶(aspartate aminotransferase，AST)变化及肾尿素氮(blood ureanitrogen，BUN)、肌酐(creatinine，CREA)变化。取处理后0.5h、1w肝、肾组织，苏木素伊红染色进行病理检测。
3.制备载基因阳离子脂质体-微泡
在自制“脂氟显”微泡基础上，以冷冻干燥法制备生物素化微泡和生物素化阳离子脂质体。将生物素化阳离子脂质体与质粒在室温下混合孵育30min，即制成载基因的生物素化阳离子脂质体。琼脂糖凝胶电泳观察不同体积比孵育质粒与阳离子脂质体的电泳情况。采用生物素-亲和素连接的办法，耦联生物素化微泡和载基因的生物素化阳离子脂质体制备载基因的新型超声微泡。激光共聚焦显微镜观察新型微泡形态及连接情况。分光光度计检测新型超声微泡载基因率。
4.超声联合新型超声微泡介导CTGF人工microRNA抗肝纤维化的实验研究
64只SD大鼠随机分为8组，第1组予生理盐水腹腔注射作为正常对照组，第2组为二甲基亚硝胺(dimethylnitrosamine，DMN)损伤建立的大鼠肝纤维化模型对照组。第3组为阴性序列对照组即载阴性序列对照质粒阳离子脂质体-微泡+超声组，第4-8组设为有效序列治疗组，分别为载基因阳离子脂质体-微泡(载基因新型微泡)+超声组、质粒+“脂氟显”微泡组+超声组、载基因新型微泡组、质粒+“脂氟显”微泡组、单纯质粒组。
治疗结束后取肝组织用HE染色、Masson染色法和Sirius胶原染色观察肝脏的病理变化。实时荧光定量PCR及Western Blot方法检测CTGF mRNA及蛋白表达水平。免疫组织化学检测TGF-β1和α-SMA蛋白表达水平。
结果
1.成功构建和筛选了靶向CTGF基因的miRNA重组质粒
构建了4个靶向CTGF基因的miRNA重组质粒，其测序结果与序列设计一致。质粒转染大鼠HSC-T6细胞后，荧光显微镜下观察到大于80%的HSC-T6细胞中观察到绿色荧光，说明质粒转染成功，而且高效表达。荧光定量PCR和Western blot结果表明，与空白对照和阴性序列质粒组比较，miRNA-CTGF-1、miRNA-CTGF-2、miRNA-CTGF-3、miRNA-CTGF-4干扰质粒均对CTGFmRNA和蛋白有明显抑制作用，其中以miRNA-CTGF-4干扰效果最为显著。
2.高机械指数诊断超声联合微泡能安全有效提高肝区微血管通透性、肝细胞的通透性以及介导基因靶向转染
肝组织内EB含量检测结果显示单纯超声组和单纯造影剂组与对照组比较没有统计学差异，超声联合微泡组与其余三组比较均有显著统计学差异。激光共聚焦显微镜下可见与其余三组比较，超声联合微泡组肝组织内可见明显增强的红色荧光，在肝细胞内可以清晰显示蓝色的胞核周围有明显的红色荧光围绕。硝酸镧电镜示踪结果显示对照组及单纯造影剂组及单纯超声组镧颗粒主要分布于肝窦毛细血管内及肝细胞间隙，未进入肝细胞内；超声联合微泡造影剂组，镧颗粒除了分布于肝窦毛细血管内及肝细胞间隙外，还可见镧颗粒进入到肝细胞内，但大部分呈颗粒状沉积于线粒体外，未进入线粒体内。超声联合微泡组处理后血清ALT和AST有一过性升高但很快恢复正常，肌酐、尿素氮虽略有波动，但其变化无统计学意义。病理结果显示超声联合微泡造影剂组处理0.5h后可见肝细胞变性，但是一周后恢复正常，对肾组织无明显影响。
3.成功制备高载基因的新型微泡载体
生物素化的阳离子脂质体平均粒径约120nm，平均表面电荷约50mV。当阳离子脂质体与质粒DNA体积比大于等于6:1时，质粒DNA可被完全阻止。激光共聚焦显微镜下观察到绿色圆形生物素化微泡周围可见红色点状的载基因生物素化阳离子脂质体。新型微泡的平均载基因率35%，普通微泡为10%。大鼠肝脏造影显示载基因阳离子脂质体-微泡能和普通微泡一样能明显增强肝脏显影。
4.超声联合新型超声微泡介导CTGF的人工microRNA抑制大鼠肝纤维化
HE染色、Masson染色和Sirius red染色显示CTGF人工microRNA能明显减轻DMN引起的大鼠肝纤维化程度。实时荧光定量PCR和Western Blot检测显示DMN模型组CTGFmRNA和蛋白水平较正常对照组明显升高，而CTGF人工microRNA处理后明显降低，以载基因新型微泡+超声组最为明显。免疫组织化学染色显示DMN模型组TGF-β1、α-SMA蛋白水平较正常对照组明显升高，CTGF人工microRNA处理后明显降低，以载基因新型微泡+超声组最为明显。
结论
1.成功构建了靶向CTGF基因的人工microRNA质粒表达载体，并筛选出对CTGF基因抑制作用最强的质粒。
2.发现高机械指数诊断超声联合微泡能安全有效提高肝区微血管通透性及肝细胞通透性。
3.生物素-亲和素系统可成功将载基因生物素化阳离子脂质体与生物素化微泡偶联起来制备出新型微泡载体，在提高微泡基因携带量的同时并不改变微泡的声学特性。
4.新型超声微泡载体联合超声介导CTGF人工microRNA能明显抑制大鼠肝纤维化程度。
Backgrounds and objectives
Hepatic fibrosis is a common pathological change in chronic liver injury characterisedby the accumulation of extracellular matrix (ECM). Following liver injury, hepatic stellatecells (HSCs) become activated and transform into myofibroblast-like cells that expressalpha-smooth muscle actin (α-SMA) and produce ECM.Transforming growth factor β1(TGF-β1) has been considered one of the most potent fibrogenic mediators to fibrosis.Because of its multiple actions, complete blockage of TGF-β1may have serious side effects.Connective tissue growth factor (CTGF) has received significant attention as a downstreameffector of the TGF-β1fibrogenic effect. CTGF not only can lead to the activation,proliferation and migration of primary HSCs, but can also promote the accumulation of ECMcomponents and inhibit their degradation. Disruption of CTGF expression has been shown toeffectively suppress the activation of HSCs and the accumulation of ECM. CTGF expressionis low or absent in normal liver tissue; however, its expression progressively increases infibrotic liver. CTGF expression is significantly correlated with the development of hepaticfibrosis. Strategies to block CTGF by small interfering RNAs (siRNA) have achievedfavourable anti-fibrotic effects for treating hepatic fibrosis.
Based on recent studies, microRNAs (miRNAs) have gained significant attention for thetreatment of hepatic fibrosis. Artificial microRNAs (amiRNAs) exploit the backbone ofnatural miRNAs to generate designed miRNAs that can efficiently silence the gene of interest.This strategy has produced highly efficient and specific gene silencing for therapeuticapplications. However, the challenge is to effectively deliver amiRNAs to the target organ.
Ultrasound-targeted microbubble destruction (UTMD) has emerged as a novel non-viralgene delivery technique because of its safety, high efficiency and local gene transfer. This method involves the attachment of genes to microbubbles, which are then injected andcirculated through blood vessels and destroyed at the target site by ultrasound insonation. Thedestruction increases capillary permeability and generates transient holes in the cellmembrane and releases the payload, which is incorporated intracellularly. Microbubbles,which consist of a lipid shell encapsulating perfluorocarbon gas. Nevertheless, thegene-loading capacity of microbubbles remains a consideration. Fortunately, studies haveshown that the loading capacity can be increased by attaching cationic liposomes to the lipidshell of microbubbles.
In this study, we utilised novel cationic liposome-bearing microbubbles combined withultrasound to transfect a plasmid-based amiRNA designed against CTGF mRNA and protein,which showed high gene-silencing efficacy in vitro, to determine whether this techniquecould efficiently suppress the expression of CTGF and exert antifibrogenic effects on hepaticfibrosis in vivo.
Methods
1. To construct microRNA eukaryotic expression vectors
Four different sequences targeting CTGF genes were designed. And also designed ascramble control sequence.The modified pre-miRNA sequence structure was derived frommurine miR-155. These sequences were annealed and ligated into five pcDNA6.2-GW/EmGFP-miR plasmids. Recombinant plasmid named miRNA-CTGF-1, miRNA-CTGF-2,miRNA-CTGF-3, miRNA-CTGF-4and miRNA-CTGF-Scramble.
Used liposome carrying method to transfect five recombinant plasmids into rat hepaticstellate cells(HSC-T6) respectively and the blank contrast only transfect the Lipofectamine2000. After24hours, observed the EmGFP expression in HSC-T6cells through invertedfluorescence microscope and evaluated there transfection efficiency.Total RNA was extractedafter incubation for48h. The protein was extracted after incubation for72h. Quantitativepolymerase chain reaction (qPCR) and western blot analysis detected the differenceexpression level of CTGF mRNA and protein in HSC-T6cells, testified to the specificityinhibitional effect of miRNA-CTGF to target genes, and specified that which recombinantplasmid had the strongest inhibitional effect and its miRNA sequence.
2. To investigate the effects of diagnostic ultrasound targeted microbubble destructionon permeability of normal liver in rats as well as its hepatic and renal toxicity.
One hundred and four rats were divided into four groups, including the group ofultrasound irradiation with frequency of1.5/3.2MHZ combined with microbubble, the groupof ultrasound irradiation only, the group of microbubble only and the control group. Thepermeability of capillary and cell membrane was detected by using Evans blue and lanthanumnitrate as tracers, respectively. Its effect on normal rat liver tissue permeability wasobserved by confocal laser microscope. Blood chemical analysed the serum ALT,AST,BUN and CREA levels.
3. To prepare DNA-loading cationic liposomes bearing-microbubbles.
Biotinylated ultrasound microbubbles and biotinylated cationic nanoliposomes wereprepared by cryochem and machine vibration．Physicochemical properties were detected.Toobtain DNA-loading liposomes, incubated the biotinylated cationic nanoliposomes and DNAplasmid at room temperature for30min. Gel retardation assay for determining the mass ratiofor the biotinylated cationic nanoliposomes complete complexation with DNA. The newDNA-loading microbubbles were made by combined microbubbles with DNA-loadingcationic nanoliposomes by biotin-avidin system. And the morphology and connect effect ofcomplex were observed by laser scanning confocal microscope. The loaded-gene ability ofthe new microbubbles were detected.
4. Inhibition of hepatic fibrosis by Artificial microRNA using cationic liposome-bearingand ultrasound
The rats were randomly divided into eight groups: the normal control group (n=8),dimethylnitrosamine (DMN, Sigma, St. Louis, MO)-induced model group (n=8), theamiScramble group (amiScramble plasmid delivered via gene-loaded cationic microbubblecombined with ultrasound, n=8) and the amiCTGF groups (n=40). The amiCTGF groups weredivided into five groups randomly (eight rats per group): amiCTGF+M++US group(amiCTGF plasmid delivered via gene-loaded microbubble combined with ultrasound),amiCTGF+M+US (amiCTGF plasmid delivered via common microbubble combined withultrasound), amiCTGF+M+group(amiCTGF plasmid delivered via gene-loaded microbubble),amiCTGF+M group(amiCTGF plasmid delivered via common microbubble) and plasmidgroup(amiCTGF plasmid only).
In the normal control group, rats received intraperitoneal saline injections for threeconsecutive days per week for up to4w. In the DMN model group, amiScramble group and amiCTGF groups, rats received intraperitoneal1%DMN (1ml/kg body weight) injections forthree consecutive days per week for up to4w. At the end of the second and third weeks, theDMN model group received a slow bolus injection of0.5ml/kg body weight of microbubbles,followed by1ml of saline to wash the tube via the caudal vein and ultrasound was applied asdescribed. At the end of the second and third weeks, the amiScrambe group and the amiCTGFgroups were given according treatments with amiScramble plasmid and amiCTGF plasmidrespectively.All rats were sacrificed4w after DMN or saline administration under generalanaesthesia. The liver tissue was quickly removed and a portion was instantly frozen in liquidnitrogen for Western blot and real-time RT-PCR analysis. Another portion was fixed in10%phosphate-buffered formalin for histopathological studies.
Results
1. Successfully constructed four MicroRNA plasmid expression vectors ofCTGFmRNAsequences，consequence of sequence analysis of those plasmids was coincidedwith computer designs. Green fluorescence could be observed in post-transfectional HSC-T6cells, which convinced that cell transfections were successful, and transfected plasmidsexpressed in cells. qPCR and Western blot showed that there was no obviously decline inCTGF mRNA and protein expression in the scambled amiRNA group(amiScamble) ascompared with the untreated HSC-T6cells group(blank) and that the four artificial microRNAtargeting CTGF groups showed different degrees of inhibitory effect, of which miRNA-CTGF-4group exhibited the strongest inhibitory effect.
2. Evans blue amount in group of ultrasound combined with microbubble was highersignificantly than that in the other three groups (P<0.01).Lanthanum nitrate-tracingtransmission electron microscope examination indicated that intracellular lanthanum could befound entering the hepatocytes in group of ultrasound combined with microbubble. Bloodchemical analysis indicated the serum ALT, AST levels was increased (P <0.01) in group ofultrasound combined with microbubble at0.5h and12h when compared to the other threegroups, but there was no significant difference at24h (P>0.01). There was no significantdifference in the serum BUN and CREA levels among the four groups after treatment. Therewas cellular swelling in liver cells in group of ultrasound combined with microbubble at0.5h,but it repaired after one week.
3. The biotinylated cationic liposomes appeared as spherical or nearly sphericalnanoparticles. The mean zeta potential was50mV. A gel retardation assay showed that thebiotinylated cationic liposomes/DNA volume ratio required to completely retain DNA in thecomplex was6:1．It showed that the green wall of biotinylated microbubbles was surroundedwith a number of red small round DNA-loading nanoliposomes. Its gene loading ability wasmuch higher than common microbubble. Contrast imaging showed that gene-loaded cationicmicrobubble could significantly enhance echo intensity of rat liver.
4. The amiCTGF treatments prevented the development of hepatic fibrosis induced byDMN, as confirmed by HE, Masson's trichrome, and Sirius red staining, of which amiCTGFdelivered via gene-loaded cationic microbubble combined with ultrasound exhibited thestrongest inhibitory effect. The expression levels of CTGF mRNA and proteins were very lowin the normal control group. But they were significantly increased in the model andamiScrambe treated groups compared with that in the normal control group (P<0.01), ofwhich amiCTGF delivered via gene-loaded cationic microbubble combined with ultrasoundexhibited the strongest inhibitory effect. However, they were markedly decreased afteramiCTGF treatment compared with those of the model and the amiScrambe treatedgroups.Moreover, compared with the model and amiScrambe treated groups, TGF-β1andα-SMA protein levels was significantly reduced after amiCTGF treatment in terms of bothquantity and intensity as determined by immunohischemistry, of which amiCTGF deliveredvia gene-loaded cationic microbubble combined with ultrasound exhibited the strongestinhibitory effect.
Conclusions
1. Successfully constructed four microRNA plasmid expression vectors of CTGFmRNA sequences; all of them inhibit CTGF mRNA and protein expression and the inhibitionrate of miRNA-CTGF-4is the most.
2. diagnostic ultrasound targeted microbubble destruction can increase the capillary andcell membrane permeability of normal liver without significant increase in hepatic and renaltoxicity.
3. A new high gene-loading microbubble was developed by conjugating gene-loadingliposomes and microbubbles, using the high affinity interaction between avidin and biotin,which had good gene carrying capacity without changing its acoustic properties．
4.Gene silencing of CTGF using amiRNAs delivered via liposome-bearing microbubbles combined with ultrasound may be a valuable specific approach for prevent hepaticfibrogenesis.
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