壓力共培養(yǎng)下HKC與HFB增殖及膠原蛋白代謝的實(shí)驗(yàn)研究
發(fā)布時(shí)間：2018-07-27 10:51
【摘要】：增生性瘢痕是人體皮膚創(chuàng)傷修復(fù)過(guò)程中的常見(jiàn)疾病,主要以成纖維細(xì)胞的過(guò)度增生及細(xì)胞外基質(zhì)的過(guò)度沉積和降解不足為特征,嚴(yán)重影響患者的身體和心理健康。對(duì)于功能部位深Ⅱ度創(chuàng)傷形成的增生性瘢痕,臨床上多采用手術(shù)方法徹底切除增生性瘢痕,移植組織工程表皮膜片覆蓋創(chuàng)面,傷口愈合后進(jìn)行壓力治療,這種治療方法具有一定的治愈效果。但是臨床上手術(shù)切除瘢痕后移植表皮及結(jié)合壓力法治療增生性瘢痕的具體機(jī)制尚不明確,有必要對(duì)其進(jìn)行更深入全面的研究。本文通過(guò)體外構(gòu)建壓力下異體表皮角質(zhì)形成細(xì)胞與真皮成纖維細(xì)胞的三維共培養(yǎng)體系,研究對(duì)細(xì)胞增殖和膠原蛋白基質(zhì)代謝的影響,從力學(xué)-生物學(xué)角度研究手術(shù)切除瘢痕后移植組織工程表皮結(jié)合壓力法治療增生性瘢痕的可能機(jī)理。本文的主要研究?jī)?nèi)容及結(jié)論如下： 1.以3%、6%、8%和10%體積的戊二醛分別交聯(lián)體積比例為3：7、1：2和1：1的2%殼聚糖與2%明膠的混合液(保持明膠溶液的體積不變),通過(guò)兩次真空冷凍干燥方法制作了12種殼聚糖-明膠支架；并檢測(cè)不同組別的殼聚糖-明膠支架的孔徑值、表觀密度、孔隙率、吸水性能、拉伸性能、壓縮性能及HaCaT細(xì)胞在支架上增殖情況的差異,研究調(diào)整殼聚糖及戊二醛的體積比例對(duì)支架性能的影響。 研究發(fā)現(xiàn)：殼聚糖-明膠支架的表觀密度、孔隙率、吸水性能、拉伸性能和壓縮性能的改變與殼聚糖和戊二醛的體積比例調(diào)整有關(guān),并選擇吸水性能、力學(xué)性能及HaCaT細(xì)胞在其上增殖良好的支架(殼聚糖與明膠的體積比例為1：1,8%體積的戊二醛制作的高2mm,直徑5mm的圓柱狀支架)作為后續(xù)實(shí)驗(yàn)培養(yǎng)皮膚細(xì)胞所使用的三維支架。 2.構(gòu)建3.4KPa氣體壓力下表皮角質(zhì)形成細(xì)胞(HKC)與真皮成纖維細(xì)胞(HFB)的三維共培養(yǎng)體系。首先采用胰酶消化法提取人包皮表皮HKC;Ⅱ型膠原酶消化法提取真皮HFB;將HKC與HFB分別以3×105/支架的密度種植于殼聚糖-明膠支架2d后,將氣-液界面誘導(dǎo)分化1d后的HKC-殼聚糖-明膠組織塊與HFB-殼聚糖-明膠組織塊共培養(yǎng)12h,應(yīng)用自制氣體壓力裝置提供3.4KPa氣體壓力,氣體壓力加載24h,并以3.4KPa壓力、無(wú)壓力單培養(yǎng)組或共培養(yǎng)組為實(shí)驗(yàn)對(duì)照組；HE染色觀察HKC與HFB在支架中的分布及生長(zhǎng)情況；MTT法測(cè)定HKC與HFB的增殖情況；分別用免疫組化方法、Q-PCR方法和ELISA方法觀察Ⅰ、Ⅲ型膠原蛋白、IL-1α和MMPP-3分別在HKC-殼聚糖-明膠與HFB-殼聚糖-明膠組織中的分布、mRNA表達(dá)及在上清液中蛋白的合成。 3.HE染色發(fā)現(xiàn)HKC與HFB均可以在殼聚糖-明膠支架上正常增殖成片；Ⅰ、Ⅲ型膠原蛋白在HKC和HFB三維組織內(nèi)呈陽(yáng)性表達(dá)；通過(guò)對(duì)比各組中HKC和HFB的增殖和膠原代謝情況發(fā)現(xiàn)：3.4KPa壓力加載可以促進(jìn)單獨(dú)培養(yǎng)的HKC增殖、膠原蛋白的合成、HKC細(xì)胞內(nèi)Ⅰ、Ⅲ型膠原蛋白mRNA的表達(dá)和蛋白的合成；抑制單獨(dú)培養(yǎng)的HFB細(xì)胞增殖、膠原蛋白的合成、HFB細(xì)胞內(nèi)Ⅰ、Ⅲ型膠原蛋白mRNA的表達(dá)和蛋白合成。HKC與HFB的無(wú)壓力共培養(yǎng)可以促進(jìn)HKC增殖和HKC細(xì)胞內(nèi)Ⅰ、Ⅲ型膠原蛋白mRNA的表達(dá),抑制HFB增殖和細(xì)胞內(nèi)Ⅰ、Ⅲ型膠原蛋白mRNA的表達(dá),且使共培養(yǎng)組分泌的膠原蛋白、Ⅰ和Ⅲ型膠原蛋白濃度均低于單獨(dú)培養(yǎng)的HFB組的濃度。3.4KPa壓力下HKC與HFB的三維共培養(yǎng)可以明顯促進(jìn)HKC增殖,Ⅰ、ⅢⅣ型膠原蛋白mRNA的表達(dá)；明顯抑制HFB增殖和對(duì)Ⅰ、Ⅲ型膠原蛋白mRNA的表達(dá),使上清液中膠原蛋白、Ⅰ和Ⅲ型膠原蛋白的合成明顯少于無(wú)壓力共培養(yǎng)組。 4.基于3.4KPa氣體壓力下HKC與HFB的三維共培養(yǎng)對(duì)細(xì)胞增殖及Ⅰ、Ⅲ型膠原蛋白mRNA表達(dá)和蛋白合成有影響的基礎(chǔ)上,關(guān)注3.4KPa氣體壓力下HKC與HFB的三維共培養(yǎng)體系中與Ⅰ、Ⅲ型膠原蛋白代謝密切相關(guān)的IL-1α和MMP-3mRNA表達(dá)和蛋白合成的變化：3.4KPa壓力可以分別促進(jìn)單獨(dú)培養(yǎng)HKC細(xì)胞內(nèi)IL-la mRNA、HFB細(xì)胞內(nèi)MMP-3mRNA的表達(dá)和蛋白的合成；HKC與HFB的三維無(wú)壓力共培養(yǎng)可以促進(jìn)HKC內(nèi)IL-1α mRNA和HFB內(nèi)MMP-3mRNA的表達(dá)；且相較于無(wú)壓力共培養(yǎng)組,3.4KPa壓力下HKC與HFB的共培養(yǎng)可以明顯促進(jìn)HKC內(nèi)IL-1α mRNA和HFB內(nèi)MMP-3mRNA的表達(dá),上清液中IL-1α和MMP-3的濃度明顯提高。 實(shí)驗(yàn)結(jié)果表明：3.4KPa壓力下HKC與HFB的三維共培養(yǎng)對(duì)HKC與HFB細(xì)胞增殖及細(xì)胞外基質(zhì)Ⅰ、Ⅲ型膠原蛋白的代謝具有一定的調(diào)控作用,且細(xì)胞因子IL-1α和基質(zhì)金屬蛋白酶MMP-3可能參與了HKC調(diào)控HFB內(nèi)Ⅰ、Ⅲ型膠原蛋白的表達(dá)和合成,這些變化有利于表皮細(xì)胞外基質(zhì)的沉積和真皮細(xì)胞外基質(zhì)的降解,有利于表皮的再上皮化和瘢痕真皮的恢復(fù)。
[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.
【學(xué)位授予單位】：太原理工大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2013
【分類(lèi)號(hào)】：R318.01
本文編號(hào)：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.
【學(xué)位授予單位】：太原理工大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2013
【分類(lèi)號(hào)】：R318.01
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