Exogenous bone morphogenetic protein-7 reduces hepatic fibrosis inSchistosoma japonicum-infected micevia transforming growth factor-β/Smad signaling

AIM: To investigate the antifibrotic effects of bone morphogenetic protein-7 (BMP-7) on Schistosoma japonicum (S. japonicum )-induced hepatic fibrosis in BALB/C mice. METHODS: Sixty BALB/C mice were randomly divided into three groups, including a control group (group A, n = 20), model group (group B, n = 20) and BMP-7 treated group (group C, n = 20). The mice in group B and group C were abdominally infected with S. japonicum cercariae to induce a schistosomal hepatic fibrosis model. The mice in group C were administered human recombinant BMP-7. Liver samples were extracted from mice sacrificed at 9 and 15 wk after modeling. Hepatic histopathological changes were assessed using Masson's staining. Transforming growth factor-beta 1 (TGF-β1), alpha-smooth muscle actin (α-SMA), phosphorylated Smad2/3 (pSmad2/3) and Smad7 protein levels and localization were measured by Western blotting and immunohistochemistry, respectively, and their mRNA expressions were detected by reverse transcriptionpolymerase chain reaction (RT-PCR). RESULTS: The schistosomal hepatic fibrosis mouse model was successfully established, as the livers of mice in group B and group C showed varying degrees of typical schistosomal hepatopathologic changes such as egg granuloma and collagen deposition. The degree of collagen deposition in group C was higher than that in group A (week 9: 22.95±6.66vs 2.02±0.76; week 15: 12.84±4.36 vs 1.74±0.80; P<0.05), but significantly lower than that in group B (week 9: 22.95±6.66 vs 34.43±6.96; week 15: 12.84±4.36 vs 18.90±5.07;P<0.05) at both time points. According to immunohistochemistry data, the expressions of α-SMA, TGF-β1 and pSmad2/3 protein in group C were higher than those in group A (α-SMA: week 9: 21.24±5.73 vs 0.33±0.20; week 15: 12.42±4.88 vs 0.34±0.27; TGF-β1: week 9: 37.00±13.74 vs 3.73±2.14; week 15: 16.71±9.80 vs 3.08±2.35; pSmad2/3: week 9: 12.92±4.81 vs 0.83±0.48; week 15: 7.87±4.09 vs 0.90±0.45; P<0.05), but significantly lower than those in group B (α-SMA: week 9: 21.24±5.73 vs 34.39±5.74; week 15: 12.42±4.88 vs 25.90±7.01; TGF-β1: week 9: 37.00±13.74 vs 55.66±14.88; week 15: 16.71±9.80 vs 37.10±12.51; pSmad2/3: week 9: 12.92±4.81 vs 19.41±6.87; week 15: 7.87±4.09vs 13.00±4.98;P<0.05) at both time points; the expression of Smad7 protein in group B was higher than that in group A and group C at week 9 (8.46±3.95 vs 1.00±0.40 and 8.46±3.95 vs 0.77±0.42; P<0.05), while there were no differences in Smad7 expression between the three groups at week 15 (1.09±0.38 vs 0.97±0.42 vs 0.89±0.39; P>0.05). Although minor discrepancies were observed, the results of RT-PCR and Western blotting were mainly consistentwith the immunohistochemical results. CONCLUSION: Exogenous BMP-7 significantly decreased the degree of hepatic fibrosis in both the acute and chronic stages of hepato-schistosomiasis, and the regulatory mechanism may involve the TGF-β/Smad signaling pathway.

Roles and regulation of bone morphogenetic protein-7 in kidney development and diseases

The gene encoding bone morphogenetic protein-7(BMP7) is expressed in the developing kidney in embryos and also in the mature organ in adults. During kidney development, expression of BMP7 is essential to determine the final number of nephrons in and proper size of the organ. The secreted BMP7 acts on the nephron progenitor cells to exert its dual functions: To maintain and expand the progenitor population and to provide them with competence to respond to differentiation cues, each relying on distinct signaling pathways. Intriguingly, in the adult organ, BMP7 has been implicated in protection against and regeneration from injury. Exogenous administration of recombinant BMP7 to animal models of kidney diseases has shown promising effects in counteracting inflammation, apoptosis and fibrosis evoked upon injury. Although the expression pattern of BMP7 has been well described, the mechanisms by which it is regulated have remained elusive and the processes by which the secretion sites of BMP7 impinge upon its functions in kidney development and diseases have not yet been assessed. Understanding the regulatory mechanisms will pave the way towards gaining better insight into the roles of BMP7, and to achieving desired control of the gene expression as a therapeutic strategy for kidney diseases.

Heterotopic ossification after the use of recombinant human bone morphogenetic protein-7

AIM To present the incidence of heterotopic ossification after the use of recombinant human bone morphogenetic protein-7(rhB MP-7) for the treatment of nonunions.METHODS Bone morphogenetic proteins(BMPs) promote bone formation by auto-induction. Recombinant human BMP-7 in combination with bone grafts was used in 84 patients for the treatment of long bone nonunions. All patients were evaluated radiographicaly for the development of heterotopic ossification during the standard assessment for the nonunion healing. In all patients(80.9%) with radiographic signs of heterotopic ossification, a CT scan was performed. Nonunion site palpation and ROM evaluation of the adjacent jointswere also carried out. Factors related to the patient(age, gender), the nonunion(location, size, chronicity, number of previous procedures, infection, surrounding tissues condition) and the surgical procedure(graft and fixation type, amount of rhB MP-7) were correlated with the development of heterotopic ossification and statistical analysis with Pearsons χ~2 test was performed.RESULTS Eighty point nine percent of the nonunions treated with rh BMP-7, healed with no need for further procedures. Heterotopic bone formation occurred in 15 of 84 patients(17.8%) and it was apparent in the routine radiologi-cal evaluation of the nonunion site, in a mean time of 5.5 mo after the rh BMP-7 application(range 3-12). The heterotopic ossification was located at the femur in 8 cases, at the tibia in 6, and at the humerus in οne patient. In 4 patients a palpable mass was present and only in one patient, with a para-articular knee nonunion treated with rhB MP-7, the size of heterotopic ossification affected the knee range of motion. All the patients with heterotopic ossification were male. Statistical analysis proved that patient's gender was the only important factor for the development of heterotopic ossification(P = 0.007). CONCLUSION Heterotopic ossification after the use of rh BMP-7 in nonunions was common but it did not compromise the final clinical outcome in most cases, and affected only male patients.

Bone morphogenetic protein-7 induced bone marrow stromal cells differentiate into neuron-like cells

Bone morphogenetic protein-7 is widely accepted as an inducer for bone marrow stem cells differentiating into osteoblasts and chondrocytes. Whether bone marrow stromal cells differentiate into neuron-like cells remains unclear. The current study examined the presence of positive cells for intermediate filament protein and microtubule associated protein-2 in the cytoplasm of bone marrow stromal cells induced by bone morphogenetic protein-7 under an inverted microscope, while no expression of glial fibrillary acidic protein was found. Reverse transcription PCR electrophoresis also revealed a positive target band for intermediate filament protein and microtubule-associated protein 2 mRNA. These results confirmed that bone morphogenetic protein-7 induces rat bone marrow stromal cells differentiating into neuron-like cells.

Construction of Adeno-associated Virus System for Human Bone Morphogenetic Protein 7 Gene

To construct the recombinant adeno-associated virus （rAAV） vector with human bone morphogenetic protein 7 （BMP7） and observe the BMP7 mRNA expression in vitro, BMP7 CDS sequence was cloned into expression plasmid pAAV-MCS of AAV Helper Free System. The recombinant plasmid was identified with enzyme digestion and sequencing. The recombinant plasmid, pAAV-RC, pHelper were co-transfected into AAV-293 cells according to the calcium phosphate-based protocol. The viral stock was collected by 4 rounds of freeze/thaw. After purified and concentrated, the recombinant virus titer was determined by dot-blot assay. HEK293 cells were transfected with the recombinant virus at different MOI, and the expression of BMP7 mRNA was detected by RT-PCR. The results showed rAAV-BMP7 was constructed and packaged successfully. The physical particle titer was 2.5×10^11 vector genomes/mL. There was different expression level of BMP7 mRNA after transfecton. These data suggested that recombinant AAV mediated a stable expression of hBMP7 mRNA in 293 cells. The AAV production method may pave the way of an effective strategy for the jaw bone defection around dental implants.

Molecular Cloning and Sequence Analysis of FullLength cDNA Encoding Human Bone Morphogenetic Protein—7

Objective:To clone the full-length human bane morphogenetic protein-7 (BMP-7 ) gene and analyse its sequence, to aid in investigation of its function and structure. Methods : Total RNA was isolated from Chinese fetal kidney by the acid gmnidinium thiocyanate phenol-chloroform method. Two overlapping segments of human BMP- 1 cDNA were obtained by reverse transcription (RT)-PCR. Following application, the two segments were ligated to each other and subcloned into POEM-T easy vector to form PEGM-T easy/hBMP-7 recombinant plasmid. Sanger dideoxy chain-termination method was used to sequence the cDNA. Results. There was 750 bp fragment obtained RT-PCR using #2 primer from 5' end of BMP-7 gene (PCR by using # 2 and # 1) ,and 540 bp fragment from 3' end was generated by KT-PCR using # 4 primer (PCR using # 3 and # 4). Full-length cDNA encoding BMP-7 was obtained by religation of two segments. When compared with hBMP-7 sequence in Gene bank (XM30619) ,our full-length BMP-7 cDNA has a G instead of a T at nucleotide 862. This change results in valine substituting for phenylalanine in the protein. Conclusion. This is the first time that BMP-7 cDNA was successfully cloned from Chinese fetal kidney. BMP-7 cDNA plays an important role in healing injuries of the osteo-articular system. This makes BMP-7 is an attractive target far various clinical applications.

Expression of mature peptide of human bone morphogenetic protein-2 in Escherichia coli

To express die mature peptide of human bone morphogenetic protein-2 in Escherichia coil. Methods: TheDNA fragment encoding the mature peptide of human bone morphogenetic protein-2 (hBMP-2m) was inserted into expression vectorpDH in which foreign gene was controlled by PRPL promoters. E. coli DH5a transformed with recombinant plasmid pDHB2m wasinduced at 42℃to express the target protein. The expressed product was partially purified and refolded, and then implanted intorat thigh muscles to assay its bone inductive activity. Results: After induction, a protein band on SDS-PAGE gel with an apparentmol. wt. of 13kD was observed to anticipate in the strain carrying pDHB2m, but not in the control. The expressed hBMP-2m accounted for 45%-60% of the total bacterial protein. The expressed product existed in a form of inclusion body. After partially purified and refolded, rhBMP-2m could induce the formation of cartilage and bone tissue heterotopically. Conclusion: The maturepeptide of human bone morphogenetic protein-2 has ben successfully expressed in E. coli and the product has ectopic bone inductive activity.

Transfection of Articular Chondrocytes with rhBMP7 Gene and Its Expression

In order to investigate the possibility of expression of exogenous gene in transduced articular chondrocytes, plasmid pcDNA3 rhBMP7 was delivered to cultured chondrocytes. Through immunohistochemical staining and RT PCR assay, the expression of rhBMP7 gene was detected. And the bioactivity of transgene expression product was detected through MTT assay as well. It was confirmed that exogenous gene could be expressed efficiently in transduced chondrocytes and the transgene expression product had obvious bioactivity. The present study provided a theoretical basis for gene therapy on the problems of articular cartilge.

芪蛭降糖胶囊对糖尿病肾病大鼠肾组织BMP-7及TGF-β_1/smads信号传导通路的影响

目的：观察芪蛭降糖胶囊对糖尿病肾病（diabetic nephropathy,DN）大鼠肾组织骨形成蛋白-7（bone morpho-genetic protein-7,BMP-7）及转化生长因子（transforming growth factor,TGF）-β1/Smads信号传导通路的影响。方法：48只清洁级雄性Wistar大鼠按体重随机抽取40只,采用切除右肾加腹腔注射链脲菌素（ streptozotocin,STZ）的方法制备DN模型,另外8只大鼠行右肾假切术。造模成功后按尿微量白蛋白（ microAlbumin,mAlb）高低,两头随机抽取分为模型组、缬沙坦对照组、芪蛭降糖胶囊低剂量组、芪蛭降糖胶囊高剂量组。成模2 d起各组给予相应浓度和剂量的药物,给药12周时观察DN大鼠尿mAlb、α1微球蛋白（α1-MG）和血清肌酐（ Scr）、尿素氮（ BUN）。然后,处死所有动物,肾组织行HE染色、PAS染色和Masson染色,观察肾组织病理变化并半定量计算肾小管损伤指数（tubulointerstitial injury index,TII）,免疫组化法检测BMP-7、TGF-β1、Smad2、Smad7在肾组织的表达。结果：给药12周后,模型组大鼠尿 mAlb、α1-MG较假手术组显著增多（ P 〈0.01）,3个治疗组DN大鼠尿mAlb、α1-MG较模型组明显减少（P〈0.01）,2个中药治疗组的上述4项指标亦较对照组降低（P〈0.05-0.01）,且呈剂量依赖性。模型组大鼠Scr、BUN亦较假手术组显著升高（P〈0.01）,对照组与模型组相比无明显变化（P〉0.05）,但2个中药治疗组较模型组明显降低（P〈0.05-0.01）。 HE染色显示,3个治疗组大鼠肾组织病理损害明显减轻,2个中药治疗组大鼠肾组织病理改善比对照组更明显；PAS染色进行TII评分发现,3个治疗组大鼠TII显著低于模型组（P〈0.01）,2个中药治疗组TII明显低于对照组（P〈0.05-0.01）；Masson染色观察发现,3个实验组大鼠肾小管间质病变明显轻于模型组,2个中药治疗组大鼠肾小管间质病变轻于对照组；免疫组化法染色并对其灰度值测定发现,3个治疗组大鼠TGF-β1、Smad2在肾组织的表达低于模型组（P〈0.01）,2个中药治疗组大鼠TGF-β1、Smad2在肾组织的表达低于对照组（P〈0.05-0.01）；3个治疗组大鼠BMP-7、Smad7在肾脏组织的表达高于模型组（P〈0.01）,2个中药治疗组大鼠BMP-7、Smad7在肾组织的表达高于对照组（P〈0.05-0.01）。结论：芪蛭降糖胶囊可能通过干预BMP-7/TGF-β1/Smads信号转导通路抑制了TGF-β1信号的细胞内转导,而对DN肾间质纤维化起到治疗作用。

BMP-7基因促进大鼠下颌牵张成骨的研究

目的:探讨骨形成蛋白-7(BMP-7)基因修饰的自体骨髓间充质干细胞(MSCs)促进大鼠下颌牵张骨痂形成的可行性。方法:选用48只雄性SD大鼠,随机分为实验和对照2组。建立大鼠下颌DO模型;于牵张结束最后1d,实验组大鼠牵张间隙内注射转染重组质粒pEGFP-BMP7的自体骨髓MSCs;而对照组大鼠注射转染pEGFP-N1空质粒的MSCs。分别于固定期第2、4、8周分3批处死大鼠,进行放射学、组织学观察,并进行骨组织形态计量学分析。结果:实验组牵张间隙内新骨形成和骨痂密度均明显高于对照组;计量学分析也显示各时间点实验组新生骨量(NBV1和NBV2)和新生骨小梁宽度(TNT)均显著高于对照组(P<0.01)。结论:基于MSCs的BMP-7exvivo基因治疗可有效促进DO新骨形成,从而缩短固定期,为临床颅颌面骨缺损的重建提供了一个极具价值的修复策略。

骨形态发生蛋白7腺病毒基因转染对骨髓基质干细胞生物学功能的影响

背景:基因转染技术正积极地应用于组织再生治疗之中,研究表明骨形态发生蛋白7具有骨诱导特性,可以有效促进成骨细胞的发育以及新骨的形成。目的:探讨骨形态发生蛋白7腺病毒基因转染对骨髓基质干细胞生物学功能的影响。方法:分离、培养羊骨髓基质干细胞,利用重组骨形态发生蛋白7腺病毒(adenovirus bone morphogenetic protein 7,Adeno-BMP7)对第2代骨髓基质干细胞进行基因转染,透射电镜观察转染后细胞超微结构。流式细胞仪检测转染后的细胞周期,Western blot检测骨形态发生蛋白7的表达,Von Kossa染色观察钙结节形态情况。取转染后第3天及相应未转染的骨髓基质干细胞制备珊瑚-细胞复合物,于裸鼠脊柱两侧背部皮下注射4周和8周后进行大体观察和组织学检测。结果与结论:体外细胞超微结构观察可见Adeno-BMP7基因转染骨髓基质干细胞存在活跃的物质合成代谢;流式细胞仪检测发现转染不会对骨髓基质干细胞的细胞周期产生明显影响;Western blot检测转染后的骨髓基质干细胞存在骨形态发生蛋白7蛋白的表达;Von Kossa染色可见转染Adeno-BMP7后的骨髓基质干细胞形成较大的钙结节。经裸鼠皮下回植实验发现,Adeno-BMP7转染后骨髓基质干细胞的成骨能力和成骨质量明显提高。以上结果表明经Adeno-BMP7转染可以有效促进骨髓基质干细胞成骨分化。

人转化生长因子β_1及骨形成蛋白7基因共表达载体的构建及对骨髓基质干细胞的定向诱导作用

目的构建人转化生长因子1β(transform ing grow th factor1β,TGF-1β)及骨形成蛋白7(bonem orphogenetic prote in 7,BM P-7)基因共表达腺病毒真核表达载体,并观察感染骨髓基质干细胞(m arrow strom a l stemce lls,M SC s)后目的基因的表达和对细胞生物学行为的影响。方法以复制缺陷的腺病毒A dE asy为基因载体,制备携带TGF-1β及BM P-7基因的高滴度腺病毒液感染人M SC s,通过免疫细胞化学、原位杂交、RT-PCR及己糖醛酸水平检测等方法鉴定外源基因的表达,分析外源性基因共表达对M SC s定向软骨分化的调控机制。结果腺病毒感染72 h后,TGF-1β和BM P-7免疫细胞化学染色可见大部份M SC s胞浆内均出现棕黄色粗颗粒,通过原位杂交检测出Ⅱ型胶原蛋白基因mRNA,感染10 d后细胞培养液中己糖醛酸含量为68.03±3.34μg/m l与感染前的53.20±3.70μg/m l明显升高有统计学意义(P<0.01)。结论成功构建了人TGF-1β及BM P-7共表达基因腺病毒表达载体,证实其在M SC s中的表达和具有向软骨细胞定向分化的诱导作用,为软骨缺损修复的局部基因治疗奠定实验基础。

重组骨形态发生蛋白-7基因转染软骨细胞的实验研究

目的 :研究外源性人重组骨形态发生蛋白 - 7(rhBMP - 7)基因在转染的软骨细胞中的表达情况。方法 :用脂质体介导法将rhBMP - 7转入兔关节软骨细胞 ,分别用免疫组化染色和逆转录 -聚合酶链式反应 (RT -PCR)检测其表达 ,同时用四甲基偶氮唑蓝微量酶反应比色法 (MTT法 )检测了表达产物的活性。结果 :48h后转染的软骨细胞有明显的 pcDNA3-rhBMP7基因表达 ,转染细胞的表达产物对正常软骨细胞的增殖有显著的促进作用。结论 :外源性rhBMP - 7基因可在软骨细胞中获得高效表达 。

重组质粒pEGFP-BMP7的构建及在大鼠骨髓间充质干细胞中的表达

目的体外构建重组质粒pEGFP_BMP7,并检测其在大鼠骨髓间充质干细胞中的表达。方法应用RT_PCR方法从大鼠肾脏中分离、扩增目的基因片段,并进行测序,随后将所得cDNA定向克隆到真核表达载体pEGFP_N1中,进行双酶切来鉴定克隆的正确性。通过脂质体介导重组pEGFP_BMP7瞬时转染大鼠骨髓间充质干细胞,确定转染效率,并通过RT_PCR、免疫细胞化学手段检测BMP7的表达。结果通过RT_PCR成功获得1.3 kb的cDNA片段,该cDNA除756 bp处有一碱基从T突变成A外,其余序列与大鼠BMP7基因完全相符。重组质粒双酶切图谱显示,BMP7 cDNA被正确插入载体中。绿色荧光蛋白在大鼠骨髓间充质干细胞中早期瞬时转染效率可达33%。转染后RT_PCR和免疫细胞化学检测证实重组pEGFP_BMP7在大鼠骨髓间充质干细胞中的表达。结论成功构建重组真核表达质粒pEGFP_BMP7,并在大鼠骨髓间充质干细胞中得到表达,有助于应用BMP7行基因治疗,促进牵张成骨、骨痂形成和修复颅颌面骨缺损。

重组人BMP-7真核表达质粒的构建及转染软骨细胞后的表达

[目的]构建重组人骨形成蛋白-7(rhBMP-7)基因真核表达质粒,转染兔关节软骨细胞,探讨外源性人rhBMP-7基因在转染软骨细胞中的表达情况及对细胞生物学性状的影响。[方法]采用PCR技术扩增rhBMP-7基因,将其插入真核表达载体pcDNA 3.1中,体外分离培养兔关节软骨细胞,然后用构建的rhBMP-7质粒转染软骨细胞,经G418筛选、免疫组化染色和逆转录PCR检测其表达,同时检测表达产物对维持软骨细胞表型的作用。[结果]经过PCR及酶切鉴定证实获得了BMP-7真核表达质粒pcDNA 3.1-rhBMP-7,通过免疫组化染色和逆转录PCR鉴定证实rhBMP-7在转染后的软骨细胞中得到了表达,其表达产物促进软骨细胞表型的维持。[结论]外源性rhBMP-7基因转染软骨细胞可以获得高效表达,并具有一定的维持软骨细胞表型的作用,为软骨组织工程的研究提供了改良的种子细胞。

腺相关病毒介导的骨形态发生蛋白7基因促种植体周骨缺损修复的实验研究

目的观察载bmp7基因的腺相关病毒(rAAV-BMP7)复合Bio-Oss的基因治疗方法对种植体周骨缺损修复的影响。方法体外构建载bmp7基因的腺相关病毒,并与Bio-Oss复合。6只雄性新西兰大白兔双侧胫骨植入种植体,并制备直径8mm、深4mm的种植体周骨缺损,A组骨缺损区填入rAAV-BMP7/Bio-Oss复合物;B组仅填入Bio-Oss;C组不充填材料。术后4、8周分期处死动物,取样进行组织学观察和形态学分析。结果A、B组骨缺损处均有新骨形成,A组较B组新骨形成更早、新生骨量更多、骨成熟程度更高(P<0.05)。结论rAAV-BMP7复合Bio-Oss较单纯植入Bio-Oss能更快、更有效地促进种植体周围骨缺损形成新骨,新骨量大且成熟度高,并能形成理想的种植体-骨结合界面。

骨形态发生蛋白-7基因转染骨髓基质细胞后的表达

为了观察重组人骨形态发生蛋白 - 7(rh BMP- 7)基因转染骨髓基质细胞 (BMSCs)后的表达及表达产物对 BM-SCs增殖的影响 ,用脂质体介导法将 rh BMP- 7基因转染 BMSCs,用逆转录 PCR技术和免疫组化 SABC法分别检测其瞬时表达和稳定表达 ;再用 3H- Td R掺入法检测基因表达产物对正常培养的 BMSCs增殖的影响。结果表明 :转基因细胞能高效表达外源基因 ,且表达时间长达 4周 ;基因表达产物能明显促进 BMSCs的增殖。

重组人骨形态发生蛋白7基因腺相关病毒载体的构建及其体外生物学活性

目的构建带有绿色荧光蛋白(GFP)标记并携带人骨形态发生蛋白7(hBMP-7)基因的无致病性重组的腺相关病毒(AAV),以其作为基因载体感染兔骨髓基质干细胞(BMSCs),体外检测病毒生物学活性及功能。方法从含有hBMP-7基因的pBluescript KS-hBMP7质粒中扩增出hBMP-7片段,构建重组骨架质粒pAAV-hBMP7-IRES-GFP。将此质粒和AAV包装质粒pAAV-RC、辅助质粒pAAV-Helper共转染AAV-293细胞,通过同源重组产生重组腺相关病毒rAAV-hBMP7-IRES-GFP。感染AAV-HT1080测定病毒滴度,应用该重组病毒感染体外培养的兔BMSCs,荧光细胞计数法检测病毒最佳感染复数,Western blot法检测BMP-7基因的体外表达,成骨诱导实验检测BMP-7的体外生物学活性。结果重组腺相关病毒表达质粒经酶切、测序验证正确。重组腺相关病毒感染滴度约为3.6×1011v.p./mL。重组病毒感染兔BMSCs的最佳感染复数为5×104v.p/细胞。Western blot结果显示重组病毒rAAV-hBMP7-IRES-GFP转染组BMSCs细胞内有BMP表达。成骨诱导实验证实rAAV-hBMP7-IRES-GFP表达的BMP体外具有良好的成骨活性。结论成功制备了rAAV-hBMP7-IRES-GFP病毒,实验获得的病毒载体滴度高、感染性好,可以满足骨组织工程的需要,为进一步体内和体外研究基因治疗促进骨愈合提供了有利的工具。

输尿管逆向注射重组腺病毒介导的BMP-7对大鼠肾间质纤维化的作用

目的:构建出具有转染后能表达活性的骨形成蛋白-7(BMP-7)的重组腺病毒,将其直接导入肾纤维化大鼠肾脏,观察其抗肾纤维化的作用。方法:无菌级雄性SD大鼠48只随机分为假手术组、单侧输尿管梗阻(UUO组)组、BMP-7治疗组,以免疫组织化学方法检测肾组织中α-SMA蛋白的表达水平,以逆转录-多聚酶链反应技术检测TGF-β1的表达水平。结果:免疫组化结果示:BMP-7组肾小管间质α-SMA表达显著减少(P<0.05)。RT-PCR结果显示:TGF-β1随梗阻时间延长而逐渐增多,第28天,有所下降,但无统计学意义。在治疗组,BMP-7组比同一识相点UUO组TGF-β1表达减少(P<0.05)。结论:BMP-7能够明显减轻肾间质纤维化,下调TGF-β1的表达,从而发挥其在单侧输尿管结扎模型中对肾脏的保护作用,延缓和抑制了肾间质纤维化的发展。