摘要
目的:增生性瘢痕是病理性瘢痕的一种,是皮肤真皮损伤愈合后瘢痕部位持续增生的一种病理现象,表现为高出正常皮肤、发红、坚硬,有时伴有瘙痒、疼痛等不适症状,不仅影响容貌和功能,还会给患者造成心理负担,而增生性瘢痕的防治是现代医学的难题之一,因此,寻找治疗和预防瘢痕增生的有效药物具有重要的意义。本课题选用著名皮肤病学专家赵炳南的经验方——黑布药膏,采用破瘀软坚之法来治疗兔耳增生性瘢痕,研究黑布药膏对兔耳增生性瘢痕动物模型成纤维细胞组织形态学、细胞增殖与凋亡及胶原合成与降解等方面的影响,从细胞、分子水平探讨黑布药膏治疗和预防增生性瘢痕的机制,为黑布药膏外用治疗增生性瘢痕提供理论和实验依据。
材料与方法:成年大耳白兔24只,随机分成3组,即正常对照组、瘢痕模型组和黑布药膏治疗组。正常对照组不施加任何处理因素,瘢痕模型组和黑布药膏治疗组进行增生性瘢痕造模,建立兔耳增生性瘢痕动物模型,21d后,造模成功,在黑布药膏治疗组瘢痕局部涂抹黑布药膏,每3d 1次,连续用药56d。在用药第2,4,6,8周分别切取两组瘢痕组织,进行指标检测。肉眼观察,在瘢痕形成和治疗过程中皮肤及瘢痕变化情况;HE染色,光镜下观察增生性瘢痕组织病理形态学改变,计算瘢痕增生指数和成纤维细胞密度;VG染色,光镜下观察增生性瘢痕组织胶原的变化,计算胶原面密度;采用免疫组化方法进行PCNA的检测,光镜下观察成纤维细胞的增殖情况,并计算PCNA阳性表达率;采用TUNNEL法进行细胞凋亡的原位检测,光镜下观察成纤维细胞的凋亡情况,并计算凋亡率;实时定量PCR(RT-PCR)法检测Bax、Bcl-2 mRNA的表达;RT-PCR法检测I、Ⅲ型胶原和基质金属蛋白酶-1 mRNA的表达;蛋白免疫印迹法(Western-blot)法检测转化生长因子-β1蛋白的表达。
结果:
1.肉眼观察,在兔耳腹侧面形成创面后,在造模后12～14d时,伤口基本愈合,并能触到硬结,造模后20～22d时,硬结更加明显,高于皮面,呈淡红色,增生范围不超过原创沿,厚度约为兔耳腹面皮肤厚度的2倍。模型组在上皮化后第六周瘢痕增生达到最高峰,厚度约为兔耳腹面皮肤厚度的3倍。黑布药膏治疗组随着给药时间的延长,瘢痕逐渐软化,体积逐渐缩小,趋于平复,色变淡。
2.光镜下观察,模型组可见真皮组织明显增生、变厚,由大量成纤维细胞、胶原组织及血管组成,胶原排列不整齐,呈结节或旋涡状分布,随时间推移,无明显变化。黑布药膏治疗组随时间推移瘢痕组织内血管、成纤维细胞逐渐减少,胶原纤维逐渐减少,呈淡红色,稀疏,排列较整齐。
3.黑布药膏对兔耳增生性瘢痕瘢痕增生指数的影响
黑布药膏治疗组较模型组瘢痕增生指数降低,从上皮化第4周开始,治疗组与模型组有显著性差异(P<0.01),并随着时间推移,瘢痕增生指数降低愈加明显。
4.黑布药膏对兔耳增生性瘢痕成纤维细胞密度的影响
模型组成纤维细胞密度明显高于正常组,与正常组比较有显著性差异(P<0.01),在上皮化后4周达到最高值,之后逐渐降低。黑布药膏可降低成纤维细胞密度,与模型组比较有显著性差异(P<0.05)。
5.黑布药膏对兔耳增生性瘢痕胶原面密度的影响
模型组胶原纤维的面密度明显高于正常组,与正常组比较有显著性差异(P<0.01),在上皮化后6周达到最高值,之后逐渐降低。黑布药膏可降低胶原纤维的面密度,从上皮化后第四周开始与模型组比较有显著性差异(P<0.01)。
6.黑布药膏对兔耳增生性瘢痕PCNA的影响
免疫组化法进行PCNA检测显示:正常皮肤表皮基底细胞层PCNA阳性表达,真皮层阳性表达较少。模型组除基底细胞PCNA阳性表达及部分棘细胞层和颗粒细胞层呈阳性表达外,真皮层见大量PCNA阳性表达细胞,与正常皮肤相比差异有显著性(P<0.01),治疗组PCNA阳性细胞较模型组明显减少,相比有显著性差异(P<0.05)。
7.黑布药膏对兔耳增生性瘢痕细胞凋亡的影响
TUNNEL法进行细胞凋亡原位检测,结果显示:在成纤维细胞和血管内皮中可见凋亡小体,即凋亡细胞。正常组皮肤中只有少量阳性细胞,瘢痕模型组上皮化2周到4周,凋亡率与正常皮肤相比无明显差异,上皮化后6周开始凋亡率降低,随时间推移凋亡率降低愈加明显,但和正常组相比没有统计学意义(P>0.05)。黑布药膏治疗组凋亡细胞较前模型组增多,真皮中见较多凋亡的成纤维细胞,而到了上皮化6周时达到凋亡高峰,至上皮化8周时凋亡率仍然持续很高。黑布药膏治疗组与模型组瘢痕凋亡率有显著性差异(P <0.01)。
8.黑布药膏对兔耳增生性瘢痕Bax mRNA表达的影响
实时定量PCR法检测Bax mRNA的表达,结果显示:与正常组比较,模型组Bax mRNA表达降低(P<0.05),并且随着上皮化时间越长,降低愈明显,从上皮化第六周开始,与正常组相比差异更加显著(P<0.01)。治疗组BaxmRNA表达较模型组明显增加,相比有显著性差异(P<0.01),并且随着上皮化时间越长,增加愈明显。
9.黑布药膏对兔耳增生性瘢痕Bcl-2mRNA表达的影响
实时定量PCR法检测Bcl-2mRNA的表达,结果显示:与正常组比较,模型组Bcl-2 mRNA表达增加(P<0.01),并且随着上皮化时间越长,增加愈明显。治疗组Bcl-2mRNA表达较模型组明显降低,从上皮化后4周开始相比有显著性差异(P<0.01),并且随着上皮化时间越长,降低愈明显。
10.黑布药膏对兔耳增生性瘢痕I型胶原mRNA表达的影响
实时定量PCR法检测I型胶原mRNA表达,结果显示:与正常组比较,模型组I型胶原mRNA表达显著升高(P<0.01),并且随着上皮化时间越长,升高愈明显。治疗组I型胶原表达较模型组降低,从上皮化后4周开始有显著性差异(P<0.01),并且随着上皮化时间越长,降低愈明显。
11.黑布药膏对兔耳增生性瘢痕Ⅲ型胶原mRAN表达的影响
实时定量PCR法检测Ⅲ型胶原mRAN表达,结果显示:模型组Ⅲ型胶原mRNA表达较正常组降低,并且随着上皮化时间越长,降低愈明显,从上皮化后6周开始,与正常组比较有显著性差异(P<0.01)。黑布药膏治疗组Ⅲ型胶原mRNA表达较模型组升高,并有显著性差异(P<0.01),并且随着上皮化时间越长,升高愈明显。
12.黑布药膏对兔耳增生性瘢痕基质金属蛋白酶-1mRNA表达的影响
实时定量PCR法检测基质金属蛋白酶-1mRNA的表达,结果显示:模型组MMP-1 mRNA表达较正常组降低,两组比较有显著性差异(P<0.01),并且随着上皮化时间越长,降低愈明显。治疗组基质金属蛋白酶-1mRNA表达较模型组明显增加,相比有显著性差异(P<0.01),并且随着上皮化时间越长,增加愈明显。
13.黑布药膏对兔耳增生性瘢痕转化生长因子-β1蛋白表达的影响
Western-blot法检测转化生长因子-β1mRNA的表达,结果显示:与正常组比较,模型组转化生长因子-β1表达显著升高(P<0.01),并且随着上皮化时间越长,升高愈明显。治疗组转化生长因子-β1表达较模型组降低,从上皮化后4周开始有显著性差异(P<0.05),从上皮化后6周开始,差异更加显著(P<0.01),并且随着上皮化时间越长,降低愈明显。
结论:
1.黑布药膏能够明显抑制成纤维细胞增殖及降低胶原纤维的含量,显著降低增生性瘢痕瘢痕增生指数、成纤维细胞密度及胶原面密度,从而抑制增生性瘢痕组织增生。
2.黑布药膏能够显著降低兔耳增生性瘢痕PCNA阳性表达率,增加细胞凋亡率,降低Bax mRNA表达,增加Bcl-2 mRNA表达,抑制增生性瘢痕成纤维细胞增殖,诱导细胞凋亡。
3.黑布药膏能够显著降低兔耳增生性瘢痕Ⅰ型胶原蛋白mRNA的表达,显著增加Ⅲ型胶原蛋白和基质金属蛋白酶-1mRNA的表达,降低转化生长因子-β1蛋白的表达,从而降低瘢痕组织胶原含量,增加胶原酶含量。
Purpose : To clarify the effect of Heibu ointment to the fibroblast histomorphology,the cell proliferation and apoptosis and the collagen synthesis of hypertrophic scar of rabbit ear animal modle,to make the mechanism clear that Heibu ointment remedy and prevent the hypertrophic scar,provide the theoretical and experimental basis for the external use of Heibu ointment. Material and method:Twenty-four white rabbits were randomly divided into normal control group, modle group and Heibu ointment treatment group .No disposition on the normal control group. The modle group and Heibu ointment treatment group were used to establish the modles of hypertrophic scar in ears.After twenty-one days,the modles were successful.Heibu ointment was plastered on the hypetrrophic scarring of Heibu ointment treatment group once every three days, it was continued fifty-six days. The material was respectively taken on the second,the fourth,the sixth and the eighth week in order to meassure the indicators.Visual inspecte the change of the skin and scars in the scar forming and therapeutic process; Observe the change of hypertrophic scars pathological morphology by HE staining technique with optical microscope,calculate the Hypertrophic Index and the fibroblastic density ; Observe the change of collagen in the scar tissue by VG staining technique calculate the collagen areal density; Measure the expression of proliferation cell nuclear antigen (PCNA) protein and apoptosis by immunohistochemistry technique, Observe the fibroblast proliferation with optical microscope, calculate PCNA positive expression rate; Situ test the apoptosis by TUNNEL technique, Observe the fibroblast apoptosis with optical microscope, calculate the apoptosis rate; Detect the expression of Bax,Bcl-2 mRNA by Real Time PCR technique; Detect the expression of type I andⅢcollagen and Matrix Metallo Proteinases-1 mRNA by Real Time PCR technique; Detect the expression of Transforming growth factor-β1 by Western-blot technique.
Results:
1.Visual inspecte,after the surface of the wound formation,the wound unioned in twelve to fourteen days.In the course of union,the hard lumps can be touched in twenty to twenty-two days,the hard lumps are rosiness and ceaseless overtop the surface of skin,the hyperplasia scop didn’t exceed the original wound scop, the thichness of the hyperplasia were two times as the mormal skin.The hyperplasia reached to the peak in the sixth weeks after epithelialization in the model control group,the thichness of the hyperplasia were three times as the mormal skin. In the Heibu ointment treatment group,as the time of treatment goes on,the scars were gradually soft,the size was gradually reduced , the hyperplasia was gradually flat and the color was gradually pale.
2. Inspecte with optical microscope, in the model group, it could be seen that the corium tissues were obvious hyperplasia and thick ,it was made up of a great number of fibroblasts,collagen tissues and vessels.The collagen ranged irregularly,the distribution was nodositas and swirls,it didn’t have obvious change as time goes on.But in the Heibu ointment treatment group,as the time of treatment goes on, the vessels, the fibroblasts and the collagens were gradually reduced.The collagens were light red,the range of the collagens was sparse and orderly.
3. The effect of Heibu Ointment on the Hypertrophic Index in the hypertrophic scarring models in rabbit ears.
Compared with the model group, the Hypertrophic Index was reduced in the Heibu ointment treatment group,from the fourth week of the epithelialization,two groups had significant difference(P<0.01).Moreover,as time goes on, the Hypertrophic Index was reduced more obviously.
4. The effect of Heibu Ointment on the fibroblastic density in the hypertrophic scarring models in rabbit ears.
The fibroblastic density of the model group was higher than the Heibu ointment treatment group, two groups had significant difference(P<0.01). On the fourth week of the epithelialization,the fibroblastic density was the highest, from then on ,it was gradually lower. The Heibu ointment could lower the fibroblastic density,compared with the model group ,they had significant difference(P<0.05)。
5. The effect of Heibu Ointment on the collagen areal density in the hypertrophic scarring models in rabbit ears.
The collagen areal density of the model group was obviously higher than the Heibu ointment treatment group, two groups had significant difference(P<0.01). On the sixth week of the epithelialization,the collagen areal density was the highest,from then on,it was gradually lower. The Heibu ointment could lower the collagen areal density,compared with the model group ,they had significant difference from the fourth week of the epithelialization(P<0.01).
6. The effect of Heibu Ointment on the PCNA in the hypertrophic scarring models in rabbit ears.
Measure the PCNA by immunohistochemistry technique. The result showed: The PCNA positive expression was in the epidermal basal cell layer of the normal skin,it was very little in the corium layer.In the model group, The PCNA positive expression was in the basal cell layer,in the granular cell layer and a great deal in the corium layer,there was significant difference between the model group an the mormal control(P<0.01). The PCNA positive expression of was lower than the Heibu ointment treatment group ,and the two group had significant difference(P<0.05).
7. The effect of Heibu Ointment on the apoptosis in the hypertrophic scarring models in rabbit ears.
Measure the apoptosis by TUNNEL technique. The result showed: The apoptotic cells could be seen in the fibroblasts and vascular endothelium.There were a few positive cells in the normal skin.The apoptosis rate of the model group didn’t have obvious difference with the normal group from the second to the fourth week of the the epithelialization. The apoptosis rate of the model group began to reduce from the sixth week of the the epithelialization, as the time goes on of the epithelialization,the reduction was more obvious,but the difference between two groups didn’t have statistical significance(P>0.05). The apoptotic cells of the Heibu ointment treatment group were more increased than the model group.It could be seen a lot of apoptotic fibroblasts,the apoptosis reached to the peak on the sixth week of the epithelialization,it was still very high on the eighth week of the epithelialization. The apoptosis rate of the Heibu ointment treatment group and the model group had significant difference(P<0.01).
8. The effect of Heibu Ointment on the expression of the Bax mRNA in the hypertrophic scarring models in rabbit ears.
Detect the expression of the Bax mRNA by Real Time PCR technique.The result showed: The expression of the Bax mRNA of the model group was lower than the normal control group ( P<0.05 ) , moreover,as the time goes on of the epithelialization,the reduction was more obvious,the difference between two groups was more significant from the sixth week of the epithelialization(P<0.01). The expression of the Bax mRNA of the Heibu ointment treatment group was higher than the model group, two groups had significant difference(P<0.01).Moreover,as the time goes on of the epithelialization,the rise was more obvious.
9. The effect of Heibu Ointment on the expression of the Bcl-2 mRNA in the hypertrophic scarring models in rabbit ears.
Detect the expression of the Bcl-2 mRNA by Real Time PCR technique.The result showed: The expression of the Bcl-2 mRNA of the model group was higher than the normal control group, two groups had significant difference(P<0.01), moreover,as the time goes on of the epithelialization,the rise was more obvious. The expression of the Bcl-2 mRNA of the Heibu ointment treatment group was lower than the model group, two groups had significant difference from the fourth week of the epithelialization ( P<0.01 ) .Moreover,as the time goes on of the epithelialization,the reduction was more obvious. 10. The effect of Heibu Ointment on the expression of the type I collagen mRNA in the hypertrophic scarring models in rabbit ears.
Detect the expression of the type I collagen mRNA by Real Time PCR technique. The result showed: The expression of the type I collagen mRNA of the model group was higher than the normal control group, two groups had significant difference(P<0.01), moreover,as the time goes on of the epithelialization,the rise was more obvious. The expression of the type I collagen mRNA of the Heibu ointment treatment group was lower than the model group, two groups had significant difference from the fourth week of the epithelialization(P<0.01).Moreover,as the time goes on of the epithelialization,the reduction was more obvious.
11. The effect of Heibu Ointment on the expression of the typeⅢcollagen mRNA in the hypertrophic scarring models in rabbit ears.
Detect the expression of the typeⅢcollagen mRNA by Real Time PCR technique.The result showed: The expression of the typeⅢcollagen mRNA of the model group was lower than the normal control group, moreover,as the time goes on of the epithelialization,the reduction was more obvious. Two groups had significant difference from the sixth week of the epithelialization(P<0.01). The expression of the typeⅢcollagen mRNA of the Heibu ointment treatment group was higher than the model group, two groups had significant difference(P<0.01).Moreover,as the time goes on of the epithelialization,the rise was more obvious.
12. The effect of Heibu Ointment on the expression of the Matrix Metallo Proteinases-1 mRNA in the hypertrophic scarring models in rabbit ears.
Detect the expression of Matrix Metallo Proteinases-1 mRNA by Real Time PCR technique.The result showed: The expression of the Matrix Metallo Proteinases-1 of the model group was lower than the normal control group, two groups had significant difference ( P<0.01 ) .Moreover,as the time goes on of the epithelialization,the reduction was more obvious. The expression of the Matrix Metallo Proteinases-1 mRNA of the Heibu ointment treatment group was higher than the model group, two groups had significant difference(P<0.01).Moreover,as the time goes on of the epithelialization,the rise was more obvious.
13. The effect of Heibu Ointment on the expression of the Transforming growth factor-β1 protein in the hypertrophic scarring models in rabbit ears.
Detect the expression of the Transforming growth factor-β1 protein by Western-blot technique.The result showed: The expression of the Transforming growth factor-β1 of the model group was higher than the normal control group, two groups had significant difference(P<0.01).Moreover,as the time goes on of the epithelialization,the rise was more obvious. The expression of the Transforming growth factor-β1 of the Heibu ointment treatment group was lower than the model group, two groups had significant difference from the fourth week of the epithelialization(P<0.05), the difference was more significant from the sixth week of the epithelialization(P<0.01).Moreover,as the time goes on of the epithelialization,the reduction was more obvious.
Conclusion:
1.Heibu ointment could obviously inhibit the fibroblast proliferation and reduce the content of the collagen synthesis,it could reduce the Hypertrophic Index, the fibroblastic density and the collagen areal density significantly,consequently it could obviously inhibit the proliferation of hypertrophic scar tissue.
2. Heibu ointment could obviously reduce the PCNA positive expression rate , increase the apoptosis rate , reduce the expression of Bax mRNA and increase the expression of Bcl-2 mRNA in the hypertrophic scar of rabbit ear. Heibu ointment could obviously restrain the fibroblast proliferation and, induce apoptosis in the hypertrophic scar.
3. Heibu ointment could obviously reduce the expression of type I andⅢcollagen mRNA, increase the expression of MMP-1 mRNA and reduce the expression of TGF-β1 in the hypertrophic scar of rabbit ear. consequently it could obviously reduce the content of the collagen and increase the content of the collagenase.
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