聚焦超聲輻照豚鼠慢性濕疹模型的生物學效應研究
發(fā)布時間：2018-07-21 10:44
【摘要】：背景 慢性濕疹是皮膚科的常見病,其特點是瘙癢明顯,易反復,局部皮膚肥厚,患者因瘙癢而搔抓,搔抓后又加重皮疹及瘙癢而形成惡性循環(huán)。聚焦超聲作為一種新興的物理治療手段已應用于婦科疾病、惡性腫瘤、外科止血等臨床治療,特別是在治療外陰白色病變中已取得了顯著療效。慢性濕疹與大部分外陰白色病變在病因、臨床表現(xiàn)及病理改變有諸多相似之處。已有研究直接或間接證實聚焦超聲可用于治療濕疹。而聚焦超聲能否成為一種既有效又方便而安全的新的物理治療方法是超聲治療皮膚病的重要研究方向之一。 目的 本實驗用2,4-二硝基氯苯(DNCB)為抗原制備豚鼠慢性濕疹模型,觀察聚焦超聲用于慢性濕疹治療的劑量及有效性和安全性,探討聚焦超聲治療慢性濕疹的可能機制,為臨床應用聚焦超聲治療慢性濕疹奠定理論基礎。 方法 本研究分為4個部分: 1.形態(tài)學和病理學變化:選擇健康豚鼠50只,分為模型組(40只)和正常組(10只),模型組每只背部剃毛2處,以2,4-二硝基氯苯(DNCB)為抗原,小劑量多次刺激豚鼠背部皮膚制備慢性濕疹模型。將40只模型隨機分為1W、2W、3W組和對照組,每組10只,分別于輻照后7d、14d取皮膚組織,HE染色病理組織學檢查。旨在探討聚焦超聲輻照豚鼠慢性濕疹模型豚鼠的皮膚病理結(jié)構(gòu)改變。 2.超微結(jié)構(gòu)改變:將32只豚鼠隨機分為4組,輻照后1d、7d、14d和對照組,用2,4-二硝基氯苯(DNCB)制備豚鼠慢性濕疹模型。輻照組以CZF型超聲治療儀對模型皮膚真皮層進行連續(xù)直線輻照,功率3W,分別于輻照后1、7和14天取材,對照組不輻照立即取材,做透射電鏡觀察。探討聚焦超聲輻照豚鼠慢性濕疹模型豚鼠的皮膚超微結(jié)構(gòu)改變。 3. IL-2和IL-4檢測:超聲輻照前,抽取正常組和模型組的外周血,輻照后7d、14d抽血,采用酶免疫(ELISA)檢測血清IL-2和IL-4含量。探討聚焦超聲輻照慢性濕疹模型豚鼠后血清IL-2和IL-4的水平變化以及其在濕疹發(fā)病機制中的作用。 4. NGF蛋白和bFGF蛋白測定:超聲輻照前,正常組和模型組取皮膚組織,輻照后7d、14d取皮膚組織,采用免疫組織化學技術(shù)研究超聲輻射豚鼠皮膚后組織中NGF蛋白和bFGF蛋白的變化。探討NGF蛋白和bFGF蛋白在慢性濕疹發(fā)病中的可能作用以及聚焦超聲治療濕疹的可能機制。 結(jié)果 1.大體觀察:模型組豚鼠皮膚表現(xiàn)為慢性濕疹樣皮損的特點。超聲輻照后即刻出現(xiàn)皮膚充血水腫加重,皮膚完整,稍硬,皮溫升高(3W2W1W),但均未見皮膚燒傷。第7天充血水腫漸退,淡紅斑,少量鱗屑,仍稍硬,皮溫正常。輻照組癥狀評分總分明顯降低,與對照組相比差異具有統(tǒng)計學意義(P0.05)。第14天基本恢復正常。 2.組織學變化:濕疹模型表皮棘層肥厚,細胞間輕度水腫,真皮大量炎癥細胞浸潤。超聲輻照后7天充血水腫消退,棘層肥厚減輕,真皮炎癥細胞浸潤減少,輻照后l4天真皮淺層少量炎癥細胞浸潤,部分膠原纖維增生,其余基本正常。真皮炎性細胞計數(shù):1W組輻照后7天和14天均與對照組無顯著差異(P0.05);2W組輻照后7天和14天均與對照組有顯著差異(P0.01);3W組輻照后7天和14天均與對照組有顯著差異(P0.001)。 3.電鏡觀察:模型組皮膚可見表皮角質(zhì)層增厚明顯,成纖維細胞線粒體腫脹、粗面內(nèi)質(zhì)網(wǎng)擴張,細胞間隙加大;輻照后第1天和7天可見血管內(nèi)皮細胞線粒體腫脹、微吞飲小泡數(shù)量多,成纖維細胞線粒體腫脹、粗面內(nèi)質(zhì)網(wǎng)擴張,但細胞間隙變小;第14天基本恢復正常。 4.豚鼠血清IL-2水平均無明顯差異。模型組豚鼠血清IL-4水平與正常組相比,明顯升高,正常組和模型組比較差異有統(tǒng)計學意義(P0.0001)。聚焦超聲能降低豚鼠升高的IL-4水平,輻照后7天, 2W、3W組與模型組有顯著差異(P0.01);輻照后14天,輻照組(1W、2W、3W)均與模型組有顯著差異(P0.001),輻照組與正常組無差異,7天和14天比較有顯著差異(P0.05)。 5. NGF蛋白和bFGF蛋白均表達于表皮和真皮附屬器的上皮細胞、成纖維細胞以及血管內(nèi)皮細胞等的胞漿和胞膜。模型組的NGF蛋白和bFGF蛋白表達明顯高于正常組(P0.001)。在聚焦超聲輻照后7和14天,1W組與對照組無顯著差異,2W組和3W組的NGF蛋白和bFGF蛋白表達明顯低于對照組(P0.05,P0.01)。輻照后14天,比較2W和3W組的NGF蛋白和bFGF蛋白表達,發(fā)現(xiàn)3W組的吸光值更加接近豚鼠正常皮膚。 結(jié)論 1.聚集超聲輻照豚鼠慢性濕疹模型可以有效地降低癥狀評分,初步結(jié)果顯示3W也是安全的,因此聚焦超聲對慢性濕疹的治療是安全和有效的,在安全劑量內(nèi),超聲輻照劑量越大,治療效果越好,聚焦超聲有利于皮膚炎癥的恢復和組織的修復。 2.通過聚焦超聲輻照豚鼠慢性濕疹模型的超微結(jié)構(gòu)觀察,聚焦超聲可用于治療濕疹,有利于微血管和膠原纖維的增生與修復,可使患處上皮細胞及真皮組織的超微結(jié)構(gòu)趨于恢復正常。 3.聚焦超聲能降低其升高的血清IL-4水平,對IL-2水平無顯著影響,可能通過降低已經(jīng)升高的IL-4水平,調(diào)節(jié)Th2分泌的IL-4之間的平衡,從而抑制Th2細胞功能發(fā)揮免疫調(diào)節(jié)作用。聚焦超聲對豚鼠慢性濕疹模型有治療作用,其作用機制可能與Th2型細胞因子有關。 4.聚焦超聲輻照豚鼠慢性濕疹模型通過超聲的生物學效應可使組織中NGF和bFGF恢復正常,進而有利于皮膚組織的修復。
[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.
【學位授予單位】：重慶醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：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.
【學位授予單位】：重慶醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R758.23
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