压力共培养下HKC与HFB增殖及胶原蛋白代谢的实验研究
发布时间：2018-07-27 10:51
【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。
[Abstract]:Hypertrophic scar is a common disease in the process of skin trauma repair. It is mainly characterized by hyperproliferation of fibroblasts and excessive deposition and degradation of extracellular matrix, which seriously affect the physical and mental health of the patients. It is not clear that the specific mechanism of the treatment of hypertrophic scar after surgical excision of cicatricial scar and combined pressure method is not clear. It is necessary to carry out more thorough and comprehensive treatment of the hypertrophic scar after surgical excision of scar. In this paper, a three-dimensional co culture system of epidermal keratinocytes and dermal fibroblasts was constructed under the pressure in vitro, and the effects on cell proliferation and collagen matrix metabolism were studied. The treatment of hypertrophic scar after surgical excision of scar after surgical excision of scar was studied from the mechanical biological angle. The main contents and conclusions of this paper are as follows:
1. the mixture of 2% chitosan and 2% gelatin with 3%, 6%, 8% and 10% volumes of 3%, 6%, 8% and 10% volumes of chitosan and 2% gelatin (keeping the volume of gelatin solution unchanged), 12 chitosan gelatin scaffolds were made by two vacuum freeze-drying methods, and the pore size of different chitosan gelatin scaffolds was detected and the apparent density was detected. Degree, porosity, water absorption, tensile properties, compression properties and the difference in the proliferation of HaCaT cells on the scaffold. The effects of the volume ratio of chitosan and glutaraldehyde on the performance of the scaffolds were studied.
It is found that the apparent density, porosity, water absorption, tensile properties and compression properties of chitosan gelatin scaffolds are related to the volume ratio adjustment of chitosan and glutaraldehyde, and choose water absorption, mechanical properties and HaCaT cells that proliferate well on it (the volume ratio of chitosan and gelatin is 1:1,8% volume. A high 2mm, diameter 5mm cylindrical scaffold made of two aldehyde) is used as a three-dimensional scaffold for subsequent skin cell culture.
2. to construct a three-dimensional co culture system of epidermal keratinocyte (HKC) and dermal fibroblast (HFB) under the pressure of 3.4KPa gas. First, the human epidermis HKC was extracted by trypsin digestion method, and HFB was extracted by type II collagenase digestion, and the density of HKC and HFB was planted on the chitosan gelatin scaffold after 2D, respectively, with the density of 3 x 105/ scaffold. The HKC- chitosan gelatin tissue block and HFB- chitosan gelatin tissue block were co cultured with 12h after 1D differentiation. The gas pressure device was used to provide 3.4KPa gas pressure, gas pressure was used to load 24h, and 3.4KPa pressure, no pressure single culture group or co culture group were used as experimental control group, and HE staining was used to observe the distribution of HKC and HFB in the scaffold. The proliferation of HKC and HFB was measured by MTT method; the distribution of type I, type III collagen, IL-1 alpha and MMPP-3 in HKC- chitosan gelatin and HFB- gelatin tissues, mRNA expression and the synthesis of protein in the supernatant were observed by immunohistochemical method, Q-PCR method and ELISA method respectively.
3.HE staining showed that both HKC and HFB could proliferate on chitosan gelatin scaffold; type I, type III collagen was positive in HKC and HFB three-dimensional tissues; by comparing the proliferation and collagen metabolism of HKC and HFB in each group, it was found that 3.4KPa pressure loading could promote the single culture of HKC proliferation, collagen synthesis, HKC. The expression of I, type III collagen mRNA and protein synthesis in cells, inhibition of the proliferation of HFB cells, synthesis of collagen, expression of type I, type III collagen mRNA in HFB cells and the pressure free co culture of.HKC and HFB in HFB cells can promote the proliferation of HKC and the expression of mRNA in type I and III collagen in HKC, and inhibit HFB. Proliferation and expression of type I, type III collagen mRNA, and the collagen protein secreted by co culture group, the concentration of type I and type III collagen was lower than that of group HFB. The three dimensional co culture of HKC and HFB under.3.4KPa pressure could obviously promote the proliferation of HKC, the expression of type I, III IV collagen mRNA, obviously inhibiting the proliferation of HFB and the proliferation of HFB. The expression of collagen type I and type III collagen made the synthesis of collagen type I and type III collagen in the supernatant significantly less than that in the non pressure co culture group mRNA.
4. based on the influence of three-dimensional co culture of HKC and HFB under 3.4KPa gas pressure on cell proliferation and mRNA expression and protein synthesis of type I, type III collagen, the expression of IL-1 A and MMP-3mRNA, which is closely related to the metabolism of type I and type III collagen, is closely related to the metabolism of HKC and HFB under 3.4KPa gas pressure. 3.4KPa pressure can promote the individual culture of IL-la mRNA in HKC cells, the expression of MMP-3mRNA in HFB cells and the synthesis of protein, and the three dimensional pressure co culture of HKC and HFB can promote the expression of IL-1 alpha mRNA in HKC and the expression within HFB, and the co culture of the pressure co culture group can be obvious compared to the non pressure co culture group. The expression of MMP-3mRNA in IL-1 alpha mRNA and HFB was promoted in HKC, and the concentration of IL-1 alpha and MMP-3 in the supernatant increased significantly.
The experimental results show that the three-dimensional co culture of HKC and HFB under 3.4KPa pressure regulates the proliferation of HKC and HFB cells and the metabolism of type I, type III collagen, and the cytokine IL-1 alpha and matrix metalloproteinase MMP-3 may be involved in the expression and synthesis of type I and III collagen in HFB, which are regulated by HKC. These changes It is beneficial to the deposition of extracellular matrix and the degradation of extracellular matrix, which is beneficial to the re epithelialization of skin and the recovery of scar skin.
【学位授予单位】：太原理工大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R318.01
本文编号：2147582
[Abstract]:Hypertrophic scar is a common disease in the process of skin trauma repair. It is mainly characterized by hyperproliferation of fibroblasts and excessive deposition and degradation of extracellular matrix, which seriously affect the physical and mental health of the patients. It is not clear that the specific mechanism of the treatment of hypertrophic scar after surgical excision of cicatricial scar and combined pressure method is not clear. It is necessary to carry out more thorough and comprehensive treatment of the hypertrophic scar after surgical excision of scar. In this paper, a three-dimensional co culture system of epidermal keratinocytes and dermal fibroblasts was constructed under the pressure in vitro, and the effects on cell proliferation and collagen matrix metabolism were studied. The treatment of hypertrophic scar after surgical excision of scar after surgical excision of scar was studied from the mechanical biological angle. The main contents and conclusions of this paper are as follows:
1. the mixture of 2% chitosan and 2% gelatin with 3%, 6%, 8% and 10% volumes of 3%, 6%, 8% and 10% volumes of chitosan and 2% gelatin (keeping the volume of gelatin solution unchanged), 12 chitosan gelatin scaffolds were made by two vacuum freeze-drying methods, and the pore size of different chitosan gelatin scaffolds was detected and the apparent density was detected. Degree, porosity, water absorption, tensile properties, compression properties and the difference in the proliferation of HaCaT cells on the scaffold. The effects of the volume ratio of chitosan and glutaraldehyde on the performance of the scaffolds were studied.
It is found that the apparent density, porosity, water absorption, tensile properties and compression properties of chitosan gelatin scaffolds are related to the volume ratio adjustment of chitosan and glutaraldehyde, and choose water absorption, mechanical properties and HaCaT cells that proliferate well on it (the volume ratio of chitosan and gelatin is 1:1,8% volume. A high 2mm, diameter 5mm cylindrical scaffold made of two aldehyde) is used as a three-dimensional scaffold for subsequent skin cell culture.
2. to construct a three-dimensional co culture system of epidermal keratinocyte (HKC) and dermal fibroblast (HFB) under the pressure of 3.4KPa gas. First, the human epidermis HKC was extracted by trypsin digestion method, and HFB was extracted by type II collagenase digestion, and the density of HKC and HFB was planted on the chitosan gelatin scaffold after 2D, respectively, with the density of 3 x 105/ scaffold. The HKC- chitosan gelatin tissue block and HFB- chitosan gelatin tissue block were co cultured with 12h after 1D differentiation. The gas pressure device was used to provide 3.4KPa gas pressure, gas pressure was used to load 24h, and 3.4KPa pressure, no pressure single culture group or co culture group were used as experimental control group, and HE staining was used to observe the distribution of HKC and HFB in the scaffold. The proliferation of HKC and HFB was measured by MTT method; the distribution of type I, type III collagen, IL-1 alpha and MMPP-3 in HKC- chitosan gelatin and HFB- gelatin tissues, mRNA expression and the synthesis of protein in the supernatant were observed by immunohistochemical method, Q-PCR method and ELISA method respectively.
3.HE staining showed that both HKC and HFB could proliferate on chitosan gelatin scaffold; type I, type III collagen was positive in HKC and HFB three-dimensional tissues; by comparing the proliferation and collagen metabolism of HKC and HFB in each group, it was found that 3.4KPa pressure loading could promote the single culture of HKC proliferation, collagen synthesis, HKC. The expression of I, type III collagen mRNA and protein synthesis in cells, inhibition of the proliferation of HFB cells, synthesis of collagen, expression of type I, type III collagen mRNA in HFB cells and the pressure free co culture of.HKC and HFB in HFB cells can promote the proliferation of HKC and the expression of mRNA in type I and III collagen in HKC, and inhibit HFB. Proliferation and expression of type I, type III collagen mRNA, and the collagen protein secreted by co culture group, the concentration of type I and type III collagen was lower than that of group HFB. The three dimensional co culture of HKC and HFB under.3.4KPa pressure could obviously promote the proliferation of HKC, the expression of type I, III IV collagen mRNA, obviously inhibiting the proliferation of HFB and the proliferation of HFB. The expression of collagen type I and type III collagen made the synthesis of collagen type I and type III collagen in the supernatant significantly less than that in the non pressure co culture group mRNA.
4. based on the influence of three-dimensional co culture of HKC and HFB under 3.4KPa gas pressure on cell proliferation and mRNA expression and protein synthesis of type I, type III collagen, the expression of IL-1 A and MMP-3mRNA, which is closely related to the metabolism of type I and type III collagen, is closely related to the metabolism of HKC and HFB under 3.4KPa gas pressure. 3.4KPa pressure can promote the individual culture of IL-la mRNA in HKC cells, the expression of MMP-3mRNA in HFB cells and the synthesis of protein, and the three dimensional pressure co culture of HKC and HFB can promote the expression of IL-1 alpha mRNA in HKC and the expression within HFB, and the co culture of the pressure co culture group can be obvious compared to the non pressure co culture group. The expression of MMP-3mRNA in IL-1 alpha mRNA and HFB was promoted in HKC, and the concentration of IL-1 alpha and MMP-3 in the supernatant increased significantly.
The experimental results show that the three-dimensional co culture of HKC and HFB under 3.4KPa pressure regulates the proliferation of HKC and HFB cells and the metabolism of type I, type III collagen, and the cytokine IL-1 alpha and matrix metalloproteinase MMP-3 may be involved in the expression and synthesis of type I and III collagen in HFB, which are regulated by HKC. These changes It is beneficial to the deposition of extracellular matrix and the degradation of extracellular matrix, which is beneficial to the re epithelialization of skin and the recovery of scar skin.
【学位授予单位】：太原理工大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R318.01
【参考文献】
相关期刊论文 前10条
1 孙文娟,蔡霞,唐胜建,刘亚红,周晓燕,马桂莲,张鹏;人皮肤角质形成细胞和成纤维细胞的原代培养和鉴定的实验研究[J];潍坊医学院学报;2004年06期
2 王德怀;钟宇;陈大夫;赵伟;张定敏;李虹;王晓;周建文;罗鹏;黄智勇;;功能部位深度烧伤的综合康复治疗320例临床观察[J];四川医学;2011年11期
3 杨军,杨光辉,刘伟,崔磊,曹谊林,钱云良;组织工程化表皮膜片的构建及其在增殖性疤痕治疗中的应用[J];上海第二医科大学学报;2004年04期
4 邓文星;张映;;实时荧光定量PCR技术综述[J];生物技术通报;2007年05期
5 ;组织工程皮肤研究的临床应用:表皮与支架材料及其他[J];中国组织工程研究与临床康复;2010年02期
6 刘飞;吴凤卿;梁智;;增生性瘢痕动物模型的研究进展[J];医学综述;2009年01期
7 ;Different roles of PKC and PKA in effect of interferon-y on proliferation and collagen synthesis of fibroblasts[J];Acta Pharmacologica Sinica;2004年10期
8 肖洪;李建福;;压应力对增生性瘢痕成纤维细胞增殖与凋亡的影响[J];中国修复重建外科杂志;2007年12期
9 张振;章一新;;增生性瘢痕治疗的研究进展[J];组织工程与重建外科杂志;2010年03期
10 周晓燕;况晓东;黄永红;徐方云;蔡震宇;王红梅;;脂氧素A_4对瘢痕成纤维细胞生物学活性的影响及机制研究[J];中国细胞生物学学报;2013年02期
相关博士学位论文 前1条
1 王国辉;后巩膜加固术治疗高度近视眼机理的力学生物学研究[D];太原理工大学;2011年
，本文编号：2147582
本文链接：https://www.wllwen.com/yixuelunwen/swyx/2147582.html
