聚焦超声辐照豚鼠慢性湿疹模型的生物学效应研究
发布时间：2018-07-21 10:44
【摘要】：背景 慢性湿疹是皮肤科的常见病,其特点是瘙痒明显,易反复,局部皮肤肥厚,患者因瘙痒而搔抓,搔抓后又加重皮疹及瘙痒而形成恶性循环。聚焦超声作为一种新兴的物理治疗手段已应用于妇科疾病、恶性肿瘤、外科止血等临床治疗,特别是在治疗外阴白色病变中已取得了显著疗效。慢性湿疹与大部分外阴白色病变在病因、临床表现及病理改变有诸多相似之处。已有研究直接或间接证实聚焦超声可用于治疗湿疹。而聚焦超声能否成为一种既有效又方便而安全的新的物理治疗方法是超声治疗皮肤病的重要研究方向之一。 目的 本实验用2,4-二硝基氯苯(DNCB)为抗原制备豚鼠慢性湿疹模型,观察聚焦超声用于慢性湿疹治疗的剂量及有效性和安全性,探讨聚焦超声治疗慢性湿疹的可能机制,为临床应用聚焦超声治疗慢性湿疹奠定理论基础。 方法 本研究分为4个部分: 1.形态学和病理学变化:选择健康豚鼠50只,分为模型组(40只)和正常组(10只),模型组每只背部剃毛2处,以2,4-二硝基氯苯(DNCB)为抗原,小剂量多次刺激豚鼠背部皮肤制备慢性湿疹模型。将40只模型随机分为1W、2W、3W组和对照组,每组10只,分别于辐照后7d、14d取皮肤组织,HE染色病理组织学检查。旨在探讨聚焦超声辐照豚鼠慢性湿疹模型豚鼠的皮肤病理结构改变。 2.超微结构改变:将32只豚鼠随机分为4组,辐照后1d、7d、14d和对照组,用2,4-二硝基氯苯(DNCB)制备豚鼠慢性湿疹模型。辐照组以CZF型超声治疗仪对模型皮肤真皮层进行连续直线辐照,功率3W,分别于辐照后1、7和14天取材,对照组不辐照立即取材,做透射电镜观察。探讨聚焦超声辐照豚鼠慢性湿疹模型豚鼠的皮肤超微结构改变。 3. IL-2和IL-4检测:超声辐照前,抽取正常组和模型组的外周血,辐照后7d、14d抽血,采用酶免疫(ELISA)检测血清IL-2和IL-4含量。探讨聚焦超声辐照慢性湿疹模型豚鼠后血清IL-2和IL-4的水平变化以及其在湿疹发病机制中的作用。 4. NGF蛋白和bFGF蛋白测定:超声辐照前,正常组和模型组取皮肤组织,辐照后7d、14d取皮肤组织,采用免疫组织化学技术研究超声辐射豚鼠皮肤后组织中NGF蛋白和bFGF蛋白的变化。探讨NGF蛋白和bFGF蛋白在慢性湿疹发病中的可能作用以及聚焦超声治疗湿疹的可能机制。 结果 1.大体观察:模型组豚鼠皮肤表现为慢性湿疹样皮损的特点。超声辐照后即刻出现皮肤充血水肿加重,皮肤完整,稍硬,皮温升高(3W2W1W),但均未见皮肤烧伤。第7天充血水肿渐退,淡红斑,少量鳞屑,仍稍硬,皮温正常。辐照组症状评分总分明显降低,与对照组相比差异具有统计学意义(P0.05)。第14天基本恢复正常。 2.组织学变化:湿疹模型表皮棘层肥厚,细胞间轻度水肿,真皮大量炎症细胞浸润。超声辐照后7天充血水肿消退,棘层肥厚减轻,真皮炎症细胞浸润减少,辐照后l4天真皮浅层少量炎症细胞浸润,部分胶原纤维增生,其余基本正常。真皮炎性细胞计数:1W组辐照后7天和14天均与对照组无显著差异(P0.05);2W组辐照后7天和14天均与对照组有显著差异(P0.01);3W组辐照后7天和14天均与对照组有显著差异(P0.001)。 3.电镜观察:模型组皮肤可见表皮角质层增厚明显,成纤维细胞线粒体肿胀、粗面内质网扩张,细胞间隙加大;辐照后第1天和7天可见血管内皮细胞线粒体肿胀、微吞饮小泡数量多,成纤维细胞线粒体肿胀、粗面内质网扩张,但细胞间隙变小;第14天基本恢复正常。 4.豚鼠血清IL-2水平均无明显差异。模型组豚鼠血清IL-4水平与正常组相比,明显升高,正常组和模型组比较差异有统计学意义(P0.0001)。聚焦超声能降低豚鼠升高的IL-4水平,辐照后7天, 2W、3W组与模型组有显著差异(P0.01);辐照后14天,辐照组(1W、2W、3W)均与模型组有显著差异(P0.001),辐照组与正常组无差异,7天和14天比较有显著差异(P0.05)。 5. NGF蛋白和bFGF蛋白均表达于表皮和真皮附属器的上皮细胞、成纤维细胞以及血管内皮细胞等的胞浆和胞膜。模型组的NGF蛋白和bFGF蛋白表达明显高于正常组(P0.001)。在聚焦超声辐照后7和14天,1W组与对照组无显著差异,2W组和3W组的NGF蛋白和bFGF蛋白表达明显低于对照组(P0.05,P0.01)。辐照后14天,比较2W和3W组的NGF蛋白和bFGF蛋白表达,发现3W组的吸光值更加接近豚鼠正常皮肤。 结论 1.聚集超声辐照豚鼠慢性湿疹模型可以有效地降低症状评分,初步结果显示3W也是安全的,因此聚焦超声对慢性湿疹的治疗是安全和有效的,在安全剂量内,超声辐照剂量越大,治疗效果越好,聚焦超声有利于皮肤炎症的恢复和组织的修复。 2.通过聚焦超声辐照豚鼠慢性湿疹模型的超微结构观察,聚焦超声可用于治疗湿疹,有利于微血管和胶原纤维的增生与修复,可使患处上皮细胞及真皮组织的超微结构趋于恢复正常。 3.聚焦超声能降低其升高的血清IL-4水平,对IL-2水平无显著影响,可能通过降低已经升高的IL-4水平,调节Th2分泌的IL-4之间的平衡,从而抑制Th2细胞功能发挥免疫调节作用。聚焦超声对豚鼠慢性湿疹模型有治疗作用,其作用机制可能与Th2型细胞因子有关。 4.聚焦超声辐照豚鼠慢性湿疹模型通过超声的生物学效应可使组织中NGF和bFGF恢复正常,进而有利于皮肤组织的修复。
[Abstract]:background
Chronic eczema is a common disease in the Department of dermatology. It is characterized by obvious itching, easy repetition, local skin hypertrophy, itching and scratching, aggravating rash and itching after scratching, and forming a vicious circle. As a new physical therapy, focused ultrasound has been applied to gynecologic diseases, malignant tumors and surgical hemostasis, especially in surgical treatment. A significant effect has been achieved in the treatment of white lesions of the vulva. There are many similarities between chronic eczema and most of the vulvar white lesions in the cause, clinical manifestation, and pathological changes. The study has been studied directly or indirectly for the treatment of eczema. Treatment is one of the important research directions of ultrasound in the treatment of dermatosis.
objective
In this experiment, 2,4- two nitrochlorobenzene (DNCB) was used as an antigen to prepare chronic eczema model in guinea pigs. The dose, effectiveness and safety of focused ultrasound used in the treatment of chronic eczema were observed. The possible mechanism of focused ultrasound in the treatment of chronic eczema was discussed, which lay a theoretical foundation for the clinical application of focused ultrasound in the treatment of chronic eczema.
Method
This study is divided into 4 parts:
1. morphological and pathological changes: 50 healthy guinea pigs were divided into model group (40 rats) and normal group (10 rats). The model group shaved 2 hair on the back, and 2,4- two nitrochlorobenzene (DNCB) was used as antigen. The model of chronic eczema was prepared by small dose of the guinea pig's back skin many times. The 40 models were randomly divided into 1W, 2W, 3W group and control group, 10 rats in each group, respectively. After irradiation, 7d, 14d took skin tissue and HE staining was examined histopathological examination. The aim of this study was to explore the skin pathological changes of guinea pig model of chronic eczema model irradiated by focused ultrasound.
2. ultrastructural changes: 32 guinea pigs were randomly divided into 4 groups. After irradiation, 1D, 7d, 14d and control group were irradiated with 2,4- two nitrochlorobenzene (DNCB) to prepare the guinea pig chronic eczema model. The irradiated group was irradiated with CZF ultrasonic instrument for the model skin dermis continuous straight line irradiation, power 3W, 1,7 and 14 days after irradiation, and the control group did not irradiate immediately. Transmission electron microscopy was used to observe the ultrastructural changes of guinea pigs with chronic eczema induced by focused ultrasound.
3. IL-2 and IL-4 tests: before ultrasonic irradiation, the peripheral blood of the normal group and the model group was extracted, 7d, 14d were extracted after irradiation, and the serum IL-2 and IL-4 contents were detected by enzyme immunoassay (ELISA). The level of serum IL-2 and IL-4 in the serum of the chronic eczema Model Guinea pigs irradiated by focused ultrasound and its role in the pathogenesis of eczema were discussed.
4. NGF protein and bFGF protein determination: before ultrasound irradiation, the skin tissue was taken from the normal group and the model group, and the skin tissue was taken by 7d, 14d after irradiation. The changes of NGF protein and bFGF protein in the tissues of the guinea pig were studied by immunohistochemistry. The possible role and focus of NGF protein and bFGF egg white in the pathogenesis of chronic eczema were discussed. The possible mechanism of ultrasound in the treatment of eczema.
Result
1. general observation: the skin of the model group was characterized by chronic eczema like skin lesions. The skin hyperemia and edema increased immediately after ultrasound irradiation, skin complete, slightly hard, and elevated skin temperature (3W2W1W), but no skin burns were found. Seventh days, congestion and edema gradually subsided, light erythema, small scale scale, still slightly hard and normal skin temperature. The symptom score of irradiation group was obvious. The difference was statistically significant (P0.05) compared with the control group, and returned to normal at fourteenth days.
2. changes of histology: hypertrophy of the epidermis of eczema, mild edema between cells and infiltration of numerous inflammatory cells in the dermis. 7 days after ultrasound irradiation, hyperemia and edema subsided, the thickness of the spinous layer reduced, the infiltration of the dermis inflammatory cells decreased, a small amount of inflammatory cells infiltrated in the superficial layer of L4 naive skin, some collagen fibers proliferated, and the rest were basically normal. Derma inflammation Cell count: there was no significant difference between the 1W group and the control group on 7 days and 14 days after irradiation (P0.05), and there was a significant difference between the group 2W and the control group (P0.01) on 7 and 14 days after irradiation (P0.01), and there was a significant difference between the 3W group and the control group after irradiation (P0.001) after 7 days and 14 days.
3. electron microscope observation: the skin of the model group showed the thickening of the cuticle, the swelling of mitochondria in the fibroblasts, the expansion of the rough endoplasmic reticulum, the enlargement of the intercellular space, and the swelling of the mitochondria in the vascular endothelial cells on the first and 7 days after irradiation, the swelling of the microvesicles, the swelling of the fibroblast cells, the expansion of the rough endoplasmic reticulum, but the small space between the cells. The fourteenth day was basically restored to normal.
There was no significant difference in serum IL-2 level between the 4. guinea pigs. The level of serum IL-4 in the model group was significantly higher than that in the normal group. The difference between the normal group and the model group was statistically significant (P0.0001). The focused ultrasound could reduce the level of IL-4 in the guinea pigs. The 2W, 3W group and the model group had significant difference (P0.01) after irradiation (P0.01), and the irradiation group (1W) after irradiation (1W) (1W). 2W (3W) was significantly different from the model group (P0.001). There was no difference between the irradiated group and the normal group, and there was a significant difference between the 7 day and the 14 day (P0.05).
5. NGF protein and bFGF protein were expressed in the epithelial cells of the epidermis and the dermal adnexa, the cytoplasm and the membrane of the fibroblasts and the vascular endothelial cells. The expression of NGF protein and bFGF protein in the model group was significantly higher than that of the normal group (P0.001). There was no significant difference between the 1W group and the control group at the 7 and 14 days after the focused ultrasound irradiation, and the NGF eggs of the 2W group and the 3W group were in the 2W and 3W groups. The expression of white and bFGF protein was significantly lower than that of the control group (P0.05, P0.01). After 14 days of irradiation, the expression of NGF and bFGF protein in 2W and 3W groups was compared, and the absorbance of 3W group was closer to the normal skin of guinea pigs.
conclusion
1. the chronic eczema model of guinea pig can effectively reduce the symptom score. The preliminary results show that 3W is also safe. Therefore, focused ultrasound is safe and effective for the treatment of chronic eczema. In the safe dose, the greater the dose of ultrasound, the better the treatment effect. The focused ultrasound is beneficial to the recovery of skin inflammation and the repair of tissue. Again.
2. the ultrastructural observation of the chronic eczema model of guinea pigs irradiated by focused ultrasound. The focused ultrasound can be used to treat eczema. It is beneficial to the proliferation and repair of microvascular and collagen fibers, and the ultrastructure of epithelial cells and dermal tissue in the affected area tends to be restored to normal.
3. focused ultrasound can reduce the level of serum IL-4, and have no significant effect on the level of IL-2. It may reduce the level of IL-4 and regulate the balance between IL-4 and the function of Th2 cells by reducing the level of Th2 secreted. The effect of focused ultrasound on the chronic humid rash model of guinea pigs may be associated with Th2 The type of cell factor is related.
4. the ultrasound induced biological effects of focal ultrasound on guinea pigs chronic eczema can restore NGF and bFGF to normal tissue, which is conducive to the repair of skin tissue.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R758.23
[Abstract]:background
Chronic eczema is a common disease in the Department of dermatology. It is characterized by obvious itching, easy repetition, local skin hypertrophy, itching and scratching, aggravating rash and itching after scratching, and forming a vicious circle. As a new physical therapy, focused ultrasound has been applied to gynecologic diseases, malignant tumors and surgical hemostasis, especially in surgical treatment. A significant effect has been achieved in the treatment of white lesions of the vulva. There are many similarities between chronic eczema and most of the vulvar white lesions in the cause, clinical manifestation, and pathological changes. The study has been studied directly or indirectly for the treatment of eczema. Treatment is one of the important research directions of ultrasound in the treatment of dermatosis.
objective
In this experiment, 2,4- two nitrochlorobenzene (DNCB) was used as an antigen to prepare chronic eczema model in guinea pigs. The dose, effectiveness and safety of focused ultrasound used in the treatment of chronic eczema were observed. The possible mechanism of focused ultrasound in the treatment of chronic eczema was discussed, which lay a theoretical foundation for the clinical application of focused ultrasound in the treatment of chronic eczema.
Method
This study is divided into 4 parts:
1. morphological and pathological changes: 50 healthy guinea pigs were divided into model group (40 rats) and normal group (10 rats). The model group shaved 2 hair on the back, and 2,4- two nitrochlorobenzene (DNCB) was used as antigen. The model of chronic eczema was prepared by small dose of the guinea pig's back skin many times. The 40 models were randomly divided into 1W, 2W, 3W group and control group, 10 rats in each group, respectively. After irradiation, 7d, 14d took skin tissue and HE staining was examined histopathological examination. The aim of this study was to explore the skin pathological changes of guinea pig model of chronic eczema model irradiated by focused ultrasound.
2. ultrastructural changes: 32 guinea pigs were randomly divided into 4 groups. After irradiation, 1D, 7d, 14d and control group were irradiated with 2,4- two nitrochlorobenzene (DNCB) to prepare the guinea pig chronic eczema model. The irradiated group was irradiated with CZF ultrasonic instrument for the model skin dermis continuous straight line irradiation, power 3W, 1,7 and 14 days after irradiation, and the control group did not irradiate immediately. Transmission electron microscopy was used to observe the ultrastructural changes of guinea pigs with chronic eczema induced by focused ultrasound.
3. IL-2 and IL-4 tests: before ultrasonic irradiation, the peripheral blood of the normal group and the model group was extracted, 7d, 14d were extracted after irradiation, and the serum IL-2 and IL-4 contents were detected by enzyme immunoassay (ELISA). The level of serum IL-2 and IL-4 in the serum of the chronic eczema Model Guinea pigs irradiated by focused ultrasound and its role in the pathogenesis of eczema were discussed.
4. NGF protein and bFGF protein determination: before ultrasound irradiation, the skin tissue was taken from the normal group and the model group, and the skin tissue was taken by 7d, 14d after irradiation. The changes of NGF protein and bFGF protein in the tissues of the guinea pig were studied by immunohistochemistry. The possible role and focus of NGF protein and bFGF egg white in the pathogenesis of chronic eczema were discussed. The possible mechanism of ultrasound in the treatment of eczema.
Result
1. general observation: the skin of the model group was characterized by chronic eczema like skin lesions. The skin hyperemia and edema increased immediately after ultrasound irradiation, skin complete, slightly hard, and elevated skin temperature (3W2W1W), but no skin burns were found. Seventh days, congestion and edema gradually subsided, light erythema, small scale scale, still slightly hard and normal skin temperature. The symptom score of irradiation group was obvious. The difference was statistically significant (P0.05) compared with the control group, and returned to normal at fourteenth days.
2. changes of histology: hypertrophy of the epidermis of eczema, mild edema between cells and infiltration of numerous inflammatory cells in the dermis. 7 days after ultrasound irradiation, hyperemia and edema subsided, the thickness of the spinous layer reduced, the infiltration of the dermis inflammatory cells decreased, a small amount of inflammatory cells infiltrated in the superficial layer of L4 naive skin, some collagen fibers proliferated, and the rest were basically normal. Derma inflammation Cell count: there was no significant difference between the 1W group and the control group on 7 days and 14 days after irradiation (P0.05), and there was a significant difference between the group 2W and the control group (P0.01) on 7 and 14 days after irradiation (P0.01), and there was a significant difference between the 3W group and the control group after irradiation (P0.001) after 7 days and 14 days.
3. electron microscope observation: the skin of the model group showed the thickening of the cuticle, the swelling of mitochondria in the fibroblasts, the expansion of the rough endoplasmic reticulum, the enlargement of the intercellular space, and the swelling of the mitochondria in the vascular endothelial cells on the first and 7 days after irradiation, the swelling of the microvesicles, the swelling of the fibroblast cells, the expansion of the rough endoplasmic reticulum, but the small space between the cells. The fourteenth day was basically restored to normal.
There was no significant difference in serum IL-2 level between the 4. guinea pigs. The level of serum IL-4 in the model group was significantly higher than that in the normal group. The difference between the normal group and the model group was statistically significant (P0.0001). The focused ultrasound could reduce the level of IL-4 in the guinea pigs. The 2W, 3W group and the model group had significant difference (P0.01) after irradiation (P0.01), and the irradiation group (1W) after irradiation (1W) (1W). 2W (3W) was significantly different from the model group (P0.001). There was no difference between the irradiated group and the normal group, and there was a significant difference between the 7 day and the 14 day (P0.05).
5. NGF protein and bFGF protein were expressed in the epithelial cells of the epidermis and the dermal adnexa, the cytoplasm and the membrane of the fibroblasts and the vascular endothelial cells. The expression of NGF protein and bFGF protein in the model group was significantly higher than that of the normal group (P0.001). There was no significant difference between the 1W group and the control group at the 7 and 14 days after the focused ultrasound irradiation, and the NGF eggs of the 2W group and the 3W group were in the 2W and 3W groups. The expression of white and bFGF protein was significantly lower than that of the control group (P0.05, P0.01). After 14 days of irradiation, the expression of NGF and bFGF protein in 2W and 3W groups was compared, and the absorbance of 3W group was closer to the normal skin of guinea pigs.
conclusion
1. the chronic eczema model of guinea pig can effectively reduce the symptom score. The preliminary results show that 3W is also safe. Therefore, focused ultrasound is safe and effective for the treatment of chronic eczema. In the safe dose, the greater the dose of ultrasound, the better the treatment effect. The focused ultrasound is beneficial to the recovery of skin inflammation and the repair of tissue. Again.
2. the ultrastructural observation of the chronic eczema model of guinea pigs irradiated by focused ultrasound. The focused ultrasound can be used to treat eczema. It is beneficial to the proliferation and repair of microvascular and collagen fibers, and the ultrastructure of epithelial cells and dermal tissue in the affected area tends to be restored to normal.
3. focused ultrasound can reduce the level of serum IL-4, and have no significant effect on the level of IL-2. It may reduce the level of IL-4 and regulate the balance between IL-4 and the function of Th2 cells by reducing the level of Th2 secreted. The effect of focused ultrasound on the chronic humid rash model of guinea pigs may be associated with Th2 The type of cell factor is related.
4. the ultrasound induced biological effects of focal ultrasound on guinea pigs chronic eczema can restore NGF and bFGF to normal tissue, which is conducive to the repair of skin tissue.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R758.23
【相似文献】
相关期刊论文 前10条
1 邬成霖,罗维丹,寇霄,陶承军;中药复方除湿汤治疗慢性湿疹的实验研究[J];中国中西医结合皮肤性病学杂志;2004年04期
2 戴庠生;;复方雄黄软膏治疗慢性湿疹61例[J];福建医药杂志;1989年06期
3 王勤,傅凤英,韩晓辉;慢性湿疹继发鳞状细胞癌一例[J];中华皮肤科杂志;1994年04期
4 徐士兴;局部封闭治疗慢性湿疹临床分析[J];皮肤病与性病;1997年03期
5 高世伟;;用“糠溜油”加热风治疗神经性皮炎及慢性湿疹[J];中国医刊;1960年06期
6 孙秋宁;手部湿疹的浅层X线治疗[J];北京医学;1986年01期
7 杨承先;苦蛇椒矾煎治湿疹104例[J];四川中医;1986年07期
8 李月玺，，张晶，翁超明;湿疹冲剂治疗急慢性湿疹90例[J];辽宁中医杂志;1996年04期
9 庞才滨 ,李永振;普爱宁乳膏治疗慢性湿疹50例疗效观察[J];广西医学;2003年06期
10 耿立东,宋步昌;中药内服外用治疗手足慢性湿疹41例[J];湖北中医杂志;2003年05期
相关会议论文 前10条
1 祝青;刁庆春;;聚焦超声辐照豚鼠慢性湿疹模型的实验研究[A];2008全国中西医结合皮肤性病学术会议论文汇编[C];2008年
2 祝青;刁庆春;;聚焦超声对慢性湿疹模型豚鼠皮肤淋巴细胞和角质形成细胞凋亡的影响[A];2008全国中西医结合皮肤性病学术会议论文汇编[C];2008年
3 祝青;刁庆春;;聚焦超声辐照豚鼠慢性湿疹模型的实验研究[A];中华医学会第14次全国皮肤性病学术年会论文汇编[C];2008年
4 李晋奇;佘姝娅;王勤;;甘草酸软膏治疗慢性湿疹的药效学[A];中国药理学会第十一次全国学术会议专刊[C];2011年
5 周英芹;於如军;;乐为联合丙酸氯倍他索霜治疗慢性湿疹的疗效观察[A];中华医学会第14次全国皮肤性病学术年会论文汇编[C];2008年
6 徐蓉;李福伦;张琳玲;宋勋;朱t熈
本文编号：2135268
本文链接：https://www.wllwen.com/yixuelunwen/pifb/2135268.html
最近更新
教材专著
