压力共培养下HKC与HFB增殖及胶原蛋白代谢的实验研究
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摘要
增生性瘢痕是人体皮肤创伤修复过程中的常见疾病,主要以成纤维细胞的过度增生及细胞外基质的过度沉积和降解不足为特征,严重影响患者的身体和心理健康。对于功能部位深Ⅱ度创伤形成的增生性瘢痕,临床上多采用手术方法彻底切除增生性瘢痕,移植组织工程表皮膜片覆盖创面,伤口愈合后进行压力治疗,这种治疗方法具有一定的治愈效果。但是临床上手术切除瘢痕后移植表皮及结合压力法治疗增生性瘢痕的具体机制尚不明确,有必要对其进行更深入全面的研究。本文通过体外构建压力下异体表皮角质形成细胞与真皮成纤维细胞的三维共培养体系,研究对细胞增殖和胶原蛋白基质代谢的影响,从力学-生物学角度研究手术切除瘢痕后移植组织工程表皮结合压力法治疗增生性瘢痕的可能机理。本文的主要研究内容及结论如下：
1.以3%、6%、8%和10%体积的戊二醛分别交联体积比例为3：7、1：2和1：1的2%壳聚糖与2%明胶的混合液(保持明胶溶液的体积不变),通过两次真空冷冻干燥方法制作了12种壳聚糖-明胶支架；并检测不同组别的壳聚糖-明胶支架的孔径值、表观密度、孔隙率、吸水性能、拉伸性能、压缩性能及HaCaT细胞在支架上增殖情况的差异,研究调整壳聚糖及戊二醛的体积比例对支架性能的影响。
研究发现：壳聚糖-明胶支架的表观密度、孔隙率、吸水性能、拉伸性能和压缩性能的改变与壳聚糖和戊二醛的体积比例调整有关,并选择吸水性能、力学性能及HaCaT细胞在其上增殖良好的支架(壳聚糖与明胶的体积比例为1：1,8%体积的戊二醛制作的高2mm,直径5mm的圆柱状支架)作为后续实验培养皮肤细胞所使用的三维支架。
2.构建3.4KPa气体压力下表皮角质形成细胞(HKC)与真皮成纤维细胞(HFB)的三维共培养体系。首先采用胰酶消化法提取人包皮表皮HKC;Ⅱ型胶原酶消化法提取真皮HFB;将HKC与HFB分别以3×105/支架的密度种植于壳聚糖-明胶支架2d后,将气-液界面诱导分化1d后的HKC-壳聚糖-明胶组织块与HFB-壳聚糖-明胶组织块共培养12h,应用自制气体压力装置提供3.4KPa气体压力,气体压力加载24h,并以3.4KPa压力、无压力单培养组或共培养组为实验对照组；HE染色观察HKC与HFB在支架中的分布及生长情况；MTT法测定HKC与HFB的增殖情况；分别用免疫组化方法、Q-PCR方法和ELISA方法观察Ⅰ、Ⅲ型胶原蛋白、IL-1α和MMPP-3分别在HKC-壳聚糖-明胶与HFB-壳聚糖-明胶组织中的分布、mRNA表达及在上清液中蛋白的合成。
3.HE染色发现HKC与HFB均可以在壳聚糖-明胶支架上正常增殖成片；Ⅰ、Ⅲ型胶原蛋白在HKC和HFB三维组织内呈阳性表达；通过对比各组中HKC和HFB的增殖和胶原代谢情况发现：3.4KPa压力加载可以促进单独培养的HKC增殖、胶原蛋白的合成、HKC细胞内Ⅰ、Ⅲ型胶原蛋白mRNA的表达和蛋白的合成；抑制单独培养的HFB细胞增殖、胶原蛋白的合成、HFB细胞内Ⅰ、Ⅲ型胶原蛋白mRNA的表达和蛋白合成。HKC与HFB的无压力共培养可以促进HKC增殖和HKC细胞内Ⅰ、Ⅲ型胶原蛋白mRNA的表达,抑制HFB增殖和细胞内Ⅰ、Ⅲ型胶原蛋白mRNA的表达,且使共培养组分泌的胶原蛋白、Ⅰ和Ⅲ型胶原蛋白浓度均低于单独培养的HFB组的浓度。3.4KPa压力下HKC与HFB的三维共培养可以明显促进HKC增殖,Ⅰ、ⅢⅣ型胶原蛋白mRNA的表达；明显抑制HFB增殖和对Ⅰ、Ⅲ型胶原蛋白mRNA的表达,使上清液中胶原蛋白、Ⅰ和Ⅲ型胶原蛋白的合成明显少于无压力共培养组。
4.基于3.4KPa气体压力下HKC与HFB的三维共培养对细胞增殖及Ⅰ、Ⅲ型胶原蛋白mRNA表达和蛋白合成有影响的基础上,关注3.4KPa气体压力下HKC与HFB的三维共培养体系中与Ⅰ、Ⅲ型胶原蛋白代谢密切相关的IL-1α和MMP-3mRNA表达和蛋白合成的变化：3.4KPa压力可以分别促进单独培养HKC细胞内IL-la mRNA、HFB细胞内MMP-3mRNA的表达和蛋白的合成；HKC与HFB的三维无压力共培养可以促进HKC内IL-1α mRNA和HFB内MMP-3mRNA的表达；且相较于无压力共培养组,3.4KPa压力下HKC与HFB的共培养可以明显促进HKC内IL-1α mRNA和HFB内MMP-3mRNA的表达,上清液中IL-1α和MMP-3的浓度明显提高。
实验结果表明：3.4KPa压力下HKC与HFB的三维共培养对HKC与HFB细胞增殖及细胞外基质Ⅰ、Ⅲ型胶原蛋白的代谢具有一定的调控作用,且细胞因子IL-1α和基质金属蛋白酶MMP-3可能参与了HKC调控HFB内Ⅰ、Ⅲ型胶原蛋白的表达和合成,这些变化有利于表皮细胞外基质的沉积和真皮细胞外基质的降解,有利于表皮的再上皮化和瘢痕真皮的恢复。
Hyperplastic scar is a common diseases formed in the process of human skin wound repairing, with fibroblast proliferated excessively and extracellular matrix deposited relentlessly and absence of degradation as characteristic, which can effect the patients' physical and mental health seriously. The combination of several treatments were used to treat hyperplastic scar in clinical application, resected the hyperplastic scar completely, transplanted skin tissue engineering to the wound using clinical operation and combining pressure treatment after taking out stitches was a common treatment to cure the hyperplastic scar formed on functional parts by wound of deep degree Ⅱ, which can gain excellent effects. However, the definite mechanism of resecting the hyperplastic scar, transplanting skin tissue engineering to the wound using clinical operation and combining pressure treatment to cure the hyperplastic scar is unclear, it is necessary to research the mechanism deeply and completely. In this paper, the system of3D coculture of xenogenous keratinocytes and fibroblasts under pressure were formed to research the effects of cell proliferation and collagen matrix metabolism. Our aim is to explore the therapeutic mechanism of resecting the hyperplastic scar, transplanting skin tissue engineering to the wound using clinical operation and combining pressure treatment to cure the hyperplastic scar from mechanics-biology view. The main content and conclusions are as following:
1.12kinds of chitosan-gelatin scaffolds were fabricated with3%、6%、8%and10%volume of glutaraldehyde as a crosslinker and mixtures of different volume of2%chitosan and2%gelatin (3:7、1:2and1:1, keeping the volume of gelatin solution unchange) by vacuum freeze-drying. Pore size, density, porosity, water absorbency, tensile and compressive properties of the scaffolds and HaCaT proliferation on the scaffolds were studied to study the effects of variation of chitosan and glutaraldehyde content on the properties of scaffolds.
It was found that porosity, density, water absorbency and mechanical properties of CG scaffolds changed with the variation of chitosan or GA content. The CG scaffolds (vr=1:1, crosslinked with8%volume of glutaraldehyde, cylindrical scaffolds with high of2mm and diameter of5mm), particularly those have excellent water absorption properties, mechanical properties and good proliferation of HaCaT cells were chose to use as3D scaffolds for growing skin cells.
2. The formation of system of3D coculture of keratinocytes and fibroblasts under pressure. Trypsin digestion method was used to extract keratinocytes form hunman foreskin; II type collagenase digestion method was used to extract fibroblasts. The HKC and HFB was planting on chitosan-gelatin scaffolds with density of3×105/scaffolds respectively for2d, and the3D HKC was cultured by air-liquid interface1d for inducing differentiation, then the3D HKC and3D HFB were cocultured in96-well plate for12h, the selfmade gas pressure application was used to offered3.4KPa pressure for24h. The groups of3.4KPa pressure, without pressure culture alone or coculture were took as control groups. HE dyeing was used to observe the distribution and growth of HKC and HFB on chitosan-gelatin scaffolds; MTT method was used to test the proliferation of HKC and HFB; Immunohistochemical method, Q-polymerase chain reaction (PCR) and ELISA method were used to observe the distribution and expression of mRNA of Ⅰ、Ⅲ type of collagen、IL-1a and MMP-3in3D organization of HKC and HFB and the concentration of supernatant fluids.
3. HKC and HFB can grow confluently on chitosan-gelatin scaffolds form HE staining pictures, and Ⅰ、Ⅲ type of collagen protein were positive expressed in HKC and HFB; through comparing the cell proliferation and collagen metabolism of HKC and HFB among ecah group it is found that:3.4KPa pressure could promote the proliferation、collagen synthesis、mRNA expression and synthesis of Ⅰ、Ⅲ type of collagen of HKC;3.4KPa pressure could restrain the proliferation、collagen synthesis、mRNA expression and synthesis of Ⅰ、Ⅲ type of collagen of HFB. The coculture of HKC and HFB without pressure could promote the proliferation、mRNA expression of Ⅰ、Ⅲ type of collagen of HKC, restrain the proliferation and mRNA expression of Ⅰ、Ⅲ type of collagen of HFB, and made the concentrations of collagen, Ⅰ and Ⅲ type of collagen were lower than those in the group of culture HFB alone. Coculture of HKC and HFB with3.4KPa pressure could promote the proliferation collagen synthesis and mRNA expression of Ⅰ、Ⅲ type of collagen of HKC obviously; and restrain the proliferation and mRNA expression of Ⅰ、Ⅲ type of collagen of HFB.
4. The IL-la and MMP-3mRNA expression and synthesis of coculture of HKC and HFB with3.4KPa pressure were tested based on the effects of coculture of HKC and HFB with3.4KPa pressure on the expression of Ⅰ、Ⅲ type of collagen mRNA and synthesis of Ⅰ、Ⅲ type of collagen.3.4KPa pressure could promote the mRNA expression and synthesis of IL-la in HKC and MMP-3in HFB; coculture of HKC and HFB without pressure could promote the mRNA expression and synthesis of IL-la in HKC and MMP-3in HFB; and coculture of HKC and HFB with3.4KPa pressure could promote mRNA expression and synthesis of IL-1α in HKC and MMP-3in HFB obviously.
It can be seen form the experimental results:there are certain regulation of coculture of HKC and HFB with3.4KPa pressure on the proliferation and extracellular matrix Ⅰ、Ⅲ type collagen metabolism of HKC and HFB, and the cytokine IL-1α and matrix metalloproteinases MMP-3may participate in the regulation of mRNA expression and synthesis of Ⅰ、Ⅲ type of collagen of HFB, which beneficial to skin extracellular matrix deposition of epidermis and extracellular matrix degradation of dermis, and advantageous to reconstruction of the skin epidermis and dermal scar disappear again.
1.以3%、6%、8%和10%体积的戊二醛分别交联体积比例为3：7、1：2和1：1的2%壳聚糖与2%明胶的混合液(保持明胶溶液的体积不变),通过两次真空冷冻干燥方法制作了12种壳聚糖-明胶支架；并检测不同组别的壳聚糖-明胶支架的孔径值、表观密度、孔隙率、吸水性能、拉伸性能、压缩性能及HaCaT细胞在支架上增殖情况的差异,研究调整壳聚糖及戊二醛的体积比例对支架性能的影响。
研究发现：壳聚糖-明胶支架的表观密度、孔隙率、吸水性能、拉伸性能和压缩性能的改变与壳聚糖和戊二醛的体积比例调整有关,并选择吸水性能、力学性能及HaCaT细胞在其上增殖良好的支架(壳聚糖与明胶的体积比例为1：1,8%体积的戊二醛制作的高2mm,直径5mm的圆柱状支架)作为后续实验培养皮肤细胞所使用的三维支架。
2.构建3.4KPa气体压力下表皮角质形成细胞(HKC)与真皮成纤维细胞(HFB)的三维共培养体系。首先采用胰酶消化法提取人包皮表皮HKC;Ⅱ型胶原酶消化法提取真皮HFB;将HKC与HFB分别以3×105/支架的密度种植于壳聚糖-明胶支架2d后,将气-液界面诱导分化1d后的HKC-壳聚糖-明胶组织块与HFB-壳聚糖-明胶组织块共培养12h,应用自制气体压力装置提供3.4KPa气体压力,气体压力加载24h,并以3.4KPa压力、无压力单培养组或共培养组为实验对照组；HE染色观察HKC与HFB在支架中的分布及生长情况；MTT法测定HKC与HFB的增殖情况；分别用免疫组化方法、Q-PCR方法和ELISA方法观察Ⅰ、Ⅲ型胶原蛋白、IL-1α和MMPP-3分别在HKC-壳聚糖-明胶与HFB-壳聚糖-明胶组织中的分布、mRNA表达及在上清液中蛋白的合成。
3.HE染色发现HKC与HFB均可以在壳聚糖-明胶支架上正常增殖成片；Ⅰ、Ⅲ型胶原蛋白在HKC和HFB三维组织内呈阳性表达；通过对比各组中HKC和HFB的增殖和胶原代谢情况发现：3.4KPa压力加载可以促进单独培养的HKC增殖、胶原蛋白的合成、HKC细胞内Ⅰ、Ⅲ型胶原蛋白mRNA的表达和蛋白的合成；抑制单独培养的HFB细胞增殖、胶原蛋白的合成、HFB细胞内Ⅰ、Ⅲ型胶原蛋白mRNA的表达和蛋白合成。HKC与HFB的无压力共培养可以促进HKC增殖和HKC细胞内Ⅰ、Ⅲ型胶原蛋白mRNA的表达,抑制HFB增殖和细胞内Ⅰ、Ⅲ型胶原蛋白mRNA的表达,且使共培养组分泌的胶原蛋白、Ⅰ和Ⅲ型胶原蛋白浓度均低于单独培养的HFB组的浓度。3.4KPa压力下HKC与HFB的三维共培养可以明显促进HKC增殖,Ⅰ、ⅢⅣ型胶原蛋白mRNA的表达；明显抑制HFB增殖和对Ⅰ、Ⅲ型胶原蛋白mRNA的表达,使上清液中胶原蛋白、Ⅰ和Ⅲ型胶原蛋白的合成明显少于无压力共培养组。
4.基于3.4KPa气体压力下HKC与HFB的三维共培养对细胞增殖及Ⅰ、Ⅲ型胶原蛋白mRNA表达和蛋白合成有影响的基础上,关注3.4KPa气体压力下HKC与HFB的三维共培养体系中与Ⅰ、Ⅲ型胶原蛋白代谢密切相关的IL-1α和MMP-3mRNA表达和蛋白合成的变化：3.4KPa压力可以分别促进单独培养HKC细胞内IL-la mRNA、HFB细胞内MMP-3mRNA的表达和蛋白的合成；HKC与HFB的三维无压力共培养可以促进HKC内IL-1α mRNA和HFB内MMP-3mRNA的表达；且相较于无压力共培养组,3.4KPa压力下HKC与HFB的共培养可以明显促进HKC内IL-1α mRNA和HFB内MMP-3mRNA的表达,上清液中IL-1α和MMP-3的浓度明显提高。
实验结果表明：3.4KPa压力下HKC与HFB的三维共培养对HKC与HFB细胞增殖及细胞外基质Ⅰ、Ⅲ型胶原蛋白的代谢具有一定的调控作用,且细胞因子IL-1α和基质金属蛋白酶MMP-3可能参与了HKC调控HFB内Ⅰ、Ⅲ型胶原蛋白的表达和合成,这些变化有利于表皮细胞外基质的沉积和真皮细胞外基质的降解,有利于表皮的再上皮化和瘢痕真皮的恢复。
Hyperplastic scar is a common diseases formed in the process of human skin wound repairing, with fibroblast proliferated excessively and extracellular matrix deposited relentlessly and absence of degradation as characteristic, which can effect the patients' physical and mental health seriously. The combination of several treatments were used to treat hyperplastic scar in clinical application, resected the hyperplastic scar completely, transplanted skin tissue engineering to the wound using clinical operation and combining pressure treatment after taking out stitches was a common treatment to cure the hyperplastic scar formed on functional parts by wound of deep degree Ⅱ, which can gain excellent effects. However, the definite mechanism of resecting the hyperplastic scar, transplanting skin tissue engineering to the wound using clinical operation and combining pressure treatment to cure the hyperplastic scar is unclear, it is necessary to research the mechanism deeply and completely. In this paper, the system of3D coculture of xenogenous keratinocytes and fibroblasts under pressure were formed to research the effects of cell proliferation and collagen matrix metabolism. Our aim is to explore the therapeutic mechanism of resecting the hyperplastic scar, transplanting skin tissue engineering to the wound using clinical operation and combining pressure treatment to cure the hyperplastic scar from mechanics-biology view. The main content and conclusions are as following:
1.12kinds of chitosan-gelatin scaffolds were fabricated with3%、6%、8%and10%volume of glutaraldehyde as a crosslinker and mixtures of different volume of2%chitosan and2%gelatin (3:7、1:2and1:1, keeping the volume of gelatin solution unchange) by vacuum freeze-drying. Pore size, density, porosity, water absorbency, tensile and compressive properties of the scaffolds and HaCaT proliferation on the scaffolds were studied to study the effects of variation of chitosan and glutaraldehyde content on the properties of scaffolds.
It was found that porosity, density, water absorbency and mechanical properties of CG scaffolds changed with the variation of chitosan or GA content. The CG scaffolds (vr=1:1, crosslinked with8%volume of glutaraldehyde, cylindrical scaffolds with high of2mm and diameter of5mm), particularly those have excellent water absorption properties, mechanical properties and good proliferation of HaCaT cells were chose to use as3D scaffolds for growing skin cells.
2. The formation of system of3D coculture of keratinocytes and fibroblasts under pressure. Trypsin digestion method was used to extract keratinocytes form hunman foreskin; II type collagenase digestion method was used to extract fibroblasts. The HKC and HFB was planting on chitosan-gelatin scaffolds with density of3×105/scaffolds respectively for2d, and the3D HKC was cultured by air-liquid interface1d for inducing differentiation, then the3D HKC and3D HFB were cocultured in96-well plate for12h, the selfmade gas pressure application was used to offered3.4KPa pressure for24h. The groups of3.4KPa pressure, without pressure culture alone or coculture were took as control groups. HE dyeing was used to observe the distribution and growth of HKC and HFB on chitosan-gelatin scaffolds; MTT method was used to test the proliferation of HKC and HFB; Immunohistochemical method, Q-polymerase chain reaction (PCR) and ELISA method were used to observe the distribution and expression of mRNA of Ⅰ、Ⅲ type of collagen、IL-1a and MMP-3in3D organization of HKC and HFB and the concentration of supernatant fluids.
3. HKC and HFB can grow confluently on chitosan-gelatin scaffolds form HE staining pictures, and Ⅰ、Ⅲ type of collagen protein were positive expressed in HKC and HFB; through comparing the cell proliferation and collagen metabolism of HKC and HFB among ecah group it is found that:3.4KPa pressure could promote the proliferation、collagen synthesis、mRNA expression and synthesis of Ⅰ、Ⅲ type of collagen of HKC;3.4KPa pressure could restrain the proliferation、collagen synthesis、mRNA expression and synthesis of Ⅰ、Ⅲ type of collagen of HFB. The coculture of HKC and HFB without pressure could promote the proliferation、mRNA expression of Ⅰ、Ⅲ type of collagen of HKC, restrain the proliferation and mRNA expression of Ⅰ、Ⅲ type of collagen of HFB, and made the concentrations of collagen, Ⅰ and Ⅲ type of collagen were lower than those in the group of culture HFB alone. Coculture of HKC and HFB with3.4KPa pressure could promote the proliferation collagen synthesis and mRNA expression of Ⅰ、Ⅲ type of collagen of HKC obviously; and restrain the proliferation and mRNA expression of Ⅰ、Ⅲ type of collagen of HFB.
4. The IL-la and MMP-3mRNA expression and synthesis of coculture of HKC and HFB with3.4KPa pressure were tested based on the effects of coculture of HKC and HFB with3.4KPa pressure on the expression of Ⅰ、Ⅲ type of collagen mRNA and synthesis of Ⅰ、Ⅲ type of collagen.3.4KPa pressure could promote the mRNA expression and synthesis of IL-la in HKC and MMP-3in HFB; coculture of HKC and HFB without pressure could promote the mRNA expression and synthesis of IL-la in HKC and MMP-3in HFB; and coculture of HKC and HFB with3.4KPa pressure could promote mRNA expression and synthesis of IL-1α in HKC and MMP-3in HFB obviously.
It can be seen form the experimental results:there are certain regulation of coculture of HKC and HFB with3.4KPa pressure on the proliferation and extracellular matrix Ⅰ、Ⅲ type collagen metabolism of HKC and HFB, and the cytokine IL-1α and matrix metalloproteinases MMP-3may participate in the regulation of mRNA expression and synthesis of Ⅰ、Ⅲ type of collagen of HFB, which beneficial to skin extracellular matrix deposition of epidermis and extracellular matrix degradation of dermis, and advantageous to reconstruction of the skin epidermis and dermal scar disappear again.
引文
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