组织工程化膀胱体内血管化的实验研究
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摘要
由于转基因细胞合成分泌的内源性蛋白表达产物活性高,能有效的同细胞表面受体结合,免疫排斥反应小,副作用小,为了探索转基因细胞在组织化膀胱体内血管化的应用,我们将VEGF165 cDNA克隆于真核表达载体pcDNA3.1(-),构建真核表达载体pcDNA3.1(-)/VEGF165;采用电转化方法将含VEGF165的真核表达质粒转染入鼠膀胱平滑肌细胞,用RT-PCR检测VEGF165基因在鼠膀胱平滑肌细胞中的表达,并用MTT法检测转染后细胞上清液中VEGF165的生物学活性。结果显示将构建的真核表达载体pcDNA3.1(-)/VEGF165转染入鼠膀胱平滑肌细胞后,VEGF的表达增高,转染后细胞上清液具有促使内皮细胞增殖的生物学活性。
为进一步了解所转染的膀胱平滑肌细胞在动物体内的促血管生成作用,我们将种植了转染VEGF的膀胱平滑肌细胞体外培养后,移植至大鼠体内,并以膀胱缺损自然愈合和单纯植入SIS为对照进行观察,于术后10周内每隔2周取标本进行组织学和免疫组化检测CD34和VEGFR2的表达,观察支架植入物的组织生长和修复情况,结果显示膀胱缺损自然愈合组的大鼠术后1周时膀胱缺损区已有膀胱移行上皮形成,第2周时新生的膀胱粘膜已覆盖缺损区。在植入SIS的两组动物中,术后1周时炎性细胞浸润明显,可见膀胱移行上皮形成;术后2周时炎性细胞减少,单纯SIS植入组和转染VEGF组均可见完整的上皮形成,两组未见明显的差异;此时已有新生毛细血管,VEGF受体的表达呈阳性,且转染VEGF组中新生毛细血管数量和VEGF受体的阳性表达细胞数高于单纯SIS植入组(P<0.05);术后4周时新生毛细血管形成较第2周时明显增多(P<0.05),但两组间新生毛细血管数和VEGF受体表达阳性细胞数未见显著性差异(P>0.05),同时两组可见少量平滑肌纤维形成;术后6周以后平滑肌形成逐渐增多,到第10周时可见明显的平滑肌束,两组间新生毛细血管数和VEGF受体表达阳性细胞数未见显著性差异(P>0.05)。
尽管文献报道制备的小肠粘膜下层中含有少量细胞生长因子,但尚不明确其在动物体内是否仍具有生物活性,为了解小肠粘膜下层在膀胱组织修复中外源性生长因子VEGF和bFGF释放,我们用ELISA法和MTT法体外测量冻干SIS在PBS孵育液中VEGF和bFGF的含量及对上皮细胞的增殖作用;用猪小肠粘膜下层(SIS)行大鼠膀胱部分修复。术后在不同时间观察大鼠膀胱修复情况,并用组织学免疫组化方法观察VEGF和bFGF的表达,单纯大鼠粘膜及部分肌层缺损组作为对照。结果显示冻干SIS在PBS孵育液中VEGF含量约为121.8±2.683ng/L; bFGF含量约为93.8±3.033ng/L,且对上皮细胞有增殖作用;组织学显示移植的SIS上可见新生毛细血管和平滑肌肌束。免疫组化显示实验组和对照组VEGF和bFGF在术后第1周均呈弱阳性表达,第2周以后实验组VEGF和bFGF表达逐渐增强,至第6周达到高峰,第8周VEGF和bFGF的表达逐渐减弱,而对照组在第1~10周VEGF和bFGF的表达均呈弱阳性,未见明显的表达高峰。可见SIS作为载体可将外源性生长因子带入大鼠体内,在SIS逐渐降解的同时释放生长因子,刺激血管生成和宿主细胞的长入和分化,但外源性生长因子的释放在SIS植入8周左右开始下降,因而如何充分利用SIS作为生长因子的载体,并使生长因子能得以持续释放,实现重建膀胱的血管化,解决细胞-基质材料移植物血液供应有待进一步探索。
Since the endogenous protein secreated by transinfected cells showed high biological activity with little immunologic rejection and side effects, which were effectively binding to surface receptor, we tried to explore better methods for the vascularization in tissue engeering bladder with transinfected cells. The VEGF165 cDNA was cloned into the eukaryotic expression vector pcDNA3.1(-). The reconstructed vector pcDNA3.1(-)/VEGF165 was transinfected into rat bladder muscular cells by electroporation. The VEGF expression in rat bladder muscular cells was determined by RT-PCR. The biological activity of the VEGF in the supernant of the transinfected cell cultures were tested by MTT method. The expression of VEGF in the transinfected bladder muscular cells of rat was improved. The proliferation of the endothelial cell was stimulated by the supernant of the transinfected cell cultures.
To understand the role of vascularization induced by transinfected bladder muscular cells in vivo, the rat’s bladders were augumentated with scaffold of small intestinal submucosa (SIS) implanted with transinfected bladder muscular cell of rat for 7 days. At the same time, another two groups of rats augumentated with SIS without transinfected cell or partial bladder mucosa and smooth muscle destroyed were used as controls. Samples were taken at regular intervals, and were detected with histological and immunohistochemistry staining against CD34 and VEGFR2 expression. In the control group without SIS transplantation, the urinary bladder transitional epithelium was observed at 1 week after operation. Neogenesis mucosa covered the destroyed area at 2 weeks. The obvious inflammatory cell infiltration and some urinary bladder transitional epithelium were observed at 1 week in the both two SIS transplanted groups (only SIS and SIS with transinfected cells). At 2 weeks after transplantation, intact epithelium was observed with no significant difference between two groups. But the numbers of neogenesis capillary and positive VEGFR2 expression cells were significantly more in the group of SIS with transinfected cells than those in only SIS group(P<0.05). At 4 weeks, the number of neogenesis capillary was significantly more than those at 2 weeks (P<0.05), but there was no significant difference between the only SIS group and SIS with transinfected cells group. Few smooth muscle fibres were observed in both groups at 4 weeks, while at 6 weeks more could be found. At 10 weeks, smooth muscle bundles were observed and there were no significant difference between the two groups of the number of neogenesis capillary and positive VEGFR2 expression cells. To understand the release of exogenous growth factors from small intestinal submuscosa (SIS) in bladder regeneration, the release of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) from SIS in vitro were evaluated by ELISA and MTT method. The defected bladder walls of rats in experimental group were repaired by porcine small intestinal submuscosa. Partial bladder mucosa and smooth muscle of the rats in control groups were destroyed. At regular intervals, the VEGF and bFGF expression were observed by histological and immunohistochemical methods. The concentration of VEGF and bFGF released in vitro from SIS in PBS solution were 121.8±2.683ng/L and 93.8±3.033ng/L respectively, and showed proliferation of vascular endothelial cell. In the SIS framework, the capillary and smooth muscle were observed followed histological evaluation. The weak expression of VEGF and bFGF in both experimental and control groups were found in the first week. Since the second week the VEGF and bFGF expression in experimental group began to increase with a peak in the 6th week, and began to decrease after 8 weeks. In the control group, the weak VEGF and bFGF expression were shown during the observation. SIS functions as a carrier for exogenous growth factors release in rat bladder regeneration. So it still needs further research on how to use the characteristics of SIS better to keep sustained growth factors level in bladder reconstruction.
为进一步了解所转染的膀胱平滑肌细胞在动物体内的促血管生成作用,我们将种植了转染VEGF的膀胱平滑肌细胞体外培养后,移植至大鼠体内,并以膀胱缺损自然愈合和单纯植入SIS为对照进行观察,于术后10周内每隔2周取标本进行组织学和免疫组化检测CD34和VEGFR2的表达,观察支架植入物的组织生长和修复情况,结果显示膀胱缺损自然愈合组的大鼠术后1周时膀胱缺损区已有膀胱移行上皮形成,第2周时新生的膀胱粘膜已覆盖缺损区。在植入SIS的两组动物中,术后1周时炎性细胞浸润明显,可见膀胱移行上皮形成;术后2周时炎性细胞减少,单纯SIS植入组和转染VEGF组均可见完整的上皮形成,两组未见明显的差异;此时已有新生毛细血管,VEGF受体的表达呈阳性,且转染VEGF组中新生毛细血管数量和VEGF受体的阳性表达细胞数高于单纯SIS植入组(P<0.05);术后4周时新生毛细血管形成较第2周时明显增多(P<0.05),但两组间新生毛细血管数和VEGF受体表达阳性细胞数未见显著性差异(P>0.05),同时两组可见少量平滑肌纤维形成;术后6周以后平滑肌形成逐渐增多,到第10周时可见明显的平滑肌束,两组间新生毛细血管数和VEGF受体表达阳性细胞数未见显著性差异(P>0.05)。
尽管文献报道制备的小肠粘膜下层中含有少量细胞生长因子,但尚不明确其在动物体内是否仍具有生物活性,为了解小肠粘膜下层在膀胱组织修复中外源性生长因子VEGF和bFGF释放,我们用ELISA法和MTT法体外测量冻干SIS在PBS孵育液中VEGF和bFGF的含量及对上皮细胞的增殖作用;用猪小肠粘膜下层(SIS)行大鼠膀胱部分修复。术后在不同时间观察大鼠膀胱修复情况,并用组织学免疫组化方法观察VEGF和bFGF的表达,单纯大鼠粘膜及部分肌层缺损组作为对照。结果显示冻干SIS在PBS孵育液中VEGF含量约为121.8±2.683ng/L; bFGF含量约为93.8±3.033ng/L,且对上皮细胞有增殖作用;组织学显示移植的SIS上可见新生毛细血管和平滑肌肌束。免疫组化显示实验组和对照组VEGF和bFGF在术后第1周均呈弱阳性表达,第2周以后实验组VEGF和bFGF表达逐渐增强,至第6周达到高峰,第8周VEGF和bFGF的表达逐渐减弱,而对照组在第1~10周VEGF和bFGF的表达均呈弱阳性,未见明显的表达高峰。可见SIS作为载体可将外源性生长因子带入大鼠体内,在SIS逐渐降解的同时释放生长因子,刺激血管生成和宿主细胞的长入和分化,但外源性生长因子的释放在SIS植入8周左右开始下降,因而如何充分利用SIS作为生长因子的载体,并使生长因子能得以持续释放,实现重建膀胱的血管化,解决细胞-基质材料移植物血液供应有待进一步探索。
Since the endogenous protein secreated by transinfected cells showed high biological activity with little immunologic rejection and side effects, which were effectively binding to surface receptor, we tried to explore better methods for the vascularization in tissue engeering bladder with transinfected cells. The VEGF165 cDNA was cloned into the eukaryotic expression vector pcDNA3.1(-). The reconstructed vector pcDNA3.1(-)/VEGF165 was transinfected into rat bladder muscular cells by electroporation. The VEGF expression in rat bladder muscular cells was determined by RT-PCR. The biological activity of the VEGF in the supernant of the transinfected cell cultures were tested by MTT method. The expression of VEGF in the transinfected bladder muscular cells of rat was improved. The proliferation of the endothelial cell was stimulated by the supernant of the transinfected cell cultures.
To understand the role of vascularization induced by transinfected bladder muscular cells in vivo, the rat’s bladders were augumentated with scaffold of small intestinal submucosa (SIS) implanted with transinfected bladder muscular cell of rat for 7 days. At the same time, another two groups of rats augumentated with SIS without transinfected cell or partial bladder mucosa and smooth muscle destroyed were used as controls. Samples were taken at regular intervals, and were detected with histological and immunohistochemistry staining against CD34 and VEGFR2 expression. In the control group without SIS transplantation, the urinary bladder transitional epithelium was observed at 1 week after operation. Neogenesis mucosa covered the destroyed area at 2 weeks. The obvious inflammatory cell infiltration and some urinary bladder transitional epithelium were observed at 1 week in the both two SIS transplanted groups (only SIS and SIS with transinfected cells). At 2 weeks after transplantation, intact epithelium was observed with no significant difference between two groups. But the numbers of neogenesis capillary and positive VEGFR2 expression cells were significantly more in the group of SIS with transinfected cells than those in only SIS group(P<0.05). At 4 weeks, the number of neogenesis capillary was significantly more than those at 2 weeks (P<0.05), but there was no significant difference between the only SIS group and SIS with transinfected cells group. Few smooth muscle fibres were observed in both groups at 4 weeks, while at 6 weeks more could be found. At 10 weeks, smooth muscle bundles were observed and there were no significant difference between the two groups of the number of neogenesis capillary and positive VEGFR2 expression cells. To understand the release of exogenous growth factors from small intestinal submuscosa (SIS) in bladder regeneration, the release of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) from SIS in vitro were evaluated by ELISA and MTT method. The defected bladder walls of rats in experimental group were repaired by porcine small intestinal submuscosa. Partial bladder mucosa and smooth muscle of the rats in control groups were destroyed. At regular intervals, the VEGF and bFGF expression were observed by histological and immunohistochemical methods. The concentration of VEGF and bFGF released in vitro from SIS in PBS solution were 121.8±2.683ng/L and 93.8±3.033ng/L respectively, and showed proliferation of vascular endothelial cell. In the SIS framework, the capillary and smooth muscle were observed followed histological evaluation. The weak expression of VEGF and bFGF in both experimental and control groups were found in the first week. Since the second week the VEGF and bFGF expression in experimental group began to increase with a peak in the 6th week, and began to decrease after 8 weeks. In the control group, the weak VEGF and bFGF expression were shown during the observation. SIS functions as a carrier for exogenous growth factors release in rat bladder regeneration. So it still needs further research on how to use the characteristics of SIS better to keep sustained growth factors level in bladder reconstruction.
引文
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62. Hidaka C, Ibarra C, Hannafin JA. Formation of vascularized meniscal tissue by combining gene therapy with tissue engineering. Tissue Eng 2002;8(1):93-105.
63. Ajoka I, Nishio R, Ikekita M et al. Establishing of heterotropic liver tissue mass with direct link to the host liver following implantation of hepatocytes transfected with vascular endothelial growth factor gene in mice. Tissue Eng 2001;7(3):335-344.
64. 吴仕和,徐迎新,阎锡蕴 纤维蛋白凝胶立体培养诱导血管样结构的形成 中华实验外科杂志 2004;21(3):337-339。
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66. 徐小鹏,王共先,袁铿等 兔膀胱平滑肌细胞体外培养初探 实用临床医学 2003;4(1):24-27.
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68. 黄晨昱 沈祖尧 血管内皮生长因子的研究及其在组织修复中的作用 中国修复重建外科杂志 2002;16(1):64-69
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71. 刘秉文,陈俊杰. 医学分子生物学,北京:中国协和医科大学出版社,2000:396.
72. Jooss K, Chirmule N. Immunity to adenovirus and adeno-associated viral vectors: implications for gene therapy. Gene Ther. 2003;10(11):955-963.
73. Thomas D, Suthanthiran M. Optimal modes and targets of gene theapy in transplantation. Immunol Rev. 2003;196:161-175.
74. Leclere PG, Panjwani A, Docherty R, et al. Effective gene delivery to adult neurons by a modified form of electroporation. J Neurosci Methods, 2005;142(1):137-143.
75. Brown MD, Schatzlein AG, Uchegbu IF. Gene dilivery with synthetic (non-viral) carriers. Int J Pharm. 2001;229 (1-2):1-21.
76. Niidome T , Huang L . Gene therapy progress and prospects : nonviral vectors. Gene Therapy , 2002 , 9 (24) ∶1647～1652.
77. 沈慧玲, 陈子兴, 王 玮, 段卫明, 傅建新 电穿孔法基因转染悬浮细胞条件的优化 苏州大学学报(医学版) 2004 ;24 (1):40-43.
78. Chen MK, Badylak SF Small bowel tissue engineering using small intestinal submucosa as a scaffold. J Surg Res. 2001;99: 352-358.
79. Kropp BP, Cheng EY, Lin HK, Zhang YY. Reliable and reproducible bladder regeneration using unseeded distal small intestinal submucosa. J Urol. 2004; 172: 1710-1718.
80. Campodonico F, Benelli R, Michelazzi A, Ognio E, Toncini C, Maffezzini M Bladder cell culture on small intestinal submucosa as bioscaffold: experimental study on engineered urothelial grafts. Eur Urol 2004; 46(4):531-537.
81. Youssif M, Shina H, Urakami S, et al. Effect of vascular endothelial growth factor on regeneration of bladder acellular matrix graft: histologic and functional evaluation. Urology 2005; 66(1): 201-207.
82. Allman AJ, McPherson TB, Badylak SF, et al. Xenogeneic extracellular matrix grafts elicit a Th2- restricted immune response. Tansplantation 2001;71(11):1631-1635.
83. Raeder RH, Badylak SF, Sheehan C, et al. Natural anti-galactose alpha 1,3 galactose antibodies delay, but do not prevent the acceptance of extracellular matrix xenograft. Transpl Immunol 2002;10(10):15-27.
84. Mcpherson TB, Liang H, Record RD, et al. Galapha (1,3) galepotope in pocine small intestinal submucosa. Tissue Eng 2000;6(3):233-245.
85. Kanematsu A, Yamamoto S, Noguchi T, Ozeki M, Tabata Y, Ogawa O. Bladder regeneration by bladder acellular matrix combined with sustained release of exogenous growth factor. J Urol. 2003;170(4 Pt 2):1633-1638.
86. Voytik-Harbin SL, Hillegonds D, Simmons C et al. Identification of extractable growth factors from small intestinal submucosa. J Cell Biochem 1997;67(4):478-481.
87. Kanematsu A, Yamamoto S, Ozeki M,et al. Collagenous matrices as release carriers of exogenous growth factors. Biomaterials. 2004 Aug;25(18):4513-20.
88. Kanematsu A, Marui A, Yamamoto S,et al. Type I collagen can function as a reservoir of basic fibroblast growth factor. J Control Release. 2004 Sep 30;99(2):281-92.
89.张路 吴宗贵 血管内皮生长因子的生物学作用及其与冠心病的关系 西医结合心脑血管病杂志 2004;2 (1):48-49。
90.张志坚,何俐 血管内皮细胞生长因子与缺血性脑血管病 中国临床康复 2004;8(7):1319-1321。