【摘要】：背景和目的： 糖尿病難愈性創(chuàng)面是包括糖尿病足和其他糖尿病創(chuàng)面在內(nèi)的，由多種原因引起的經(jīng)久不愈的創(chuàng)面，是最嚴(yán)重、花費(fèi)最高的糖尿病慢性并發(fā)癥之一，嚴(yán)重的糖尿病足可導(dǎo)致截肢、甚至死亡。調(diào)查顯示，我國(guó)約有30%的非創(chuàng)傷性截肢是由于糖尿病足引發(fā)。糖尿病難愈性創(chuàng)面發(fā)生發(fā)展機(jī)制及護(hù)理是臨床研究的難點(diǎn)和熱點(diǎn)。 TGF-β1/Smads信號(hào)轉(zhuǎn)導(dǎo)通路是糖尿病難愈性創(chuàng)面修復(fù)中重要的信號(hào)轉(zhuǎn)導(dǎo)通路之一。TGF-β1是介導(dǎo)組織硬化和間質(zhì)纖維化的關(guān)鍵細(xì)胞因子，參與了細(xì)胞分化、增殖、粘附、血管生成、炎癥反應(yīng)和創(chuàng)面愈合等過(guò)程，還能促成成纖維細(xì)胞的遷移和增殖，趨化、刺激成纖維細(xì)胞生成細(xì)胞外基質(zhì)。研究顯示，糖尿病難愈性創(chuàng)面的形成與創(chuàng)面TGF-β1表達(dá)的絕對(duì)或相對(duì)不足有關(guān)。近年，弱酸環(huán)境對(duì)難愈性創(chuàng)面的愈合有促進(jìn)作用的臨床個(gè)案已有少量報(bào)道。我們認(rèn)為其機(jī)制除了弱酸環(huán)境對(duì)創(chuàng)面微生物有一定的抑制作用外，還可能與其能刺激創(chuàng)面Tgf-β1mRNA的表達(dá)有關(guān)，這可能是其促進(jìn)創(chuàng)面愈合的重要機(jī)制之一，值得深入研究。 4%硼酸溶液作為臨床常見的消毒劑，具有弱酸性和較低的細(xì)胞毒性，多用于皮膚、粘膜及傷口的消毒處置，，有部分臨床研究也曾運(yùn)用硼酸對(duì)下肢淺表潰瘍、褥瘡等創(chuàng)面進(jìn)行處理并取得了較好效果。本研究因此進(jìn)行了硼酸消毒液促進(jìn)糖尿病難愈性創(chuàng)面愈合的護(hù)理效用及其相關(guān)機(jī)制的實(shí)驗(yàn)研究，以期為臨床糖尿病難愈性創(chuàng)面護(hù)理措施的細(xì)化和修訂提供更科學(xué)的參考依據(jù)。 材料與方法： 1.動(dòng)物分組與模型建立：8周齡雄性C57BL/6小鼠30只，購(gòu)買并飼養(yǎng)于第三軍醫(yī)大學(xué)實(shí)驗(yàn)動(dòng)物中心SPF級(jí)動(dòng)物實(shí)驗(yàn)室，隨機(jī)分為糖尿病組與正常對(duì)照組，各15只。每組中又分為3天、7天、14天三個(gè)時(shí)相點(diǎn)（每個(gè)時(shí)相點(diǎn)5只）。糖尿病組小鼠以鏈脲佐菌素誘導(dǎo)建模，角膜環(huán)鉆制作小鼠背部創(chuàng)面。 2.每日1次，在同一時(shí)段分別以0.9%生理鹽水（A處理）、4%硼酸溶液（B處理）、0.5%碘伏+3%過(guò)氧化氫溶液（C處理）對(duì)不同組別小鼠創(chuàng)面進(jìn)行清洗換藥，觀察記錄創(chuàng)面結(jié)痂情況、愈合情況及創(chuàng)面大小，并拍照。 3.在不同時(shí)相點(diǎn)用Trizol提取小鼠創(chuàng)面組織的總RNA，在檢測(cè)RNA濃度、RT-PCR反應(yīng)后，獲得cDNA。 4.采用SYBR Green Q-PCR法對(duì)上述創(chuàng)面組織的cDNA進(jìn)行Tgf-β1、Smad4、Collagen I的mRNA相對(duì)表達(dá)量檢測(cè)。 結(jié)果： 1.硼酸對(duì)創(chuàng)面愈合率的影響 糖尿病硼酸處理組各時(shí)相點(diǎn)創(chuàng)面愈合率較其余處理組的創(chuàng)面愈合率提高明顯（P＜0.05）。 2.硼酸對(duì)創(chuàng)面Tgf-β1mRNA表達(dá)的影響 正常小鼠硼酸處理組3天時(shí)Tgf-β1mRNA的表達(dá)均較其余處理組明顯增高（P＜0.05）；糖尿病小鼠硼酸處理組在傷后3天、14天時(shí)其Tgf-β1mRNA的表達(dá)較其余處理組有顯著升高（P＜0.01）。 3.硼酸對(duì)創(chuàng)面Smad4mRNA表達(dá)的影響 正常小鼠硼酸處理組傷后14天時(shí)其Smad4mRNA表達(dá)低于其余處理組（P＜0.05）；糖尿病小鼠傷后14天生理鹽水組Smad4mRNA的表達(dá)明顯低于碘伏+雙氧水處理組（P＜0.05）。 4.硼酸對(duì)創(chuàng)面Collagen I mRNA表達(dá)的影響 糖尿病小鼠和正常小鼠在傷后3天時(shí)均表現(xiàn)為硼酸處理組和碘伏+雙氧水處理組的Collagen I mRNA的表達(dá)高于生理鹽水組（P＜0.05）；14天時(shí)，糖尿病小鼠硼酸組和生理鹽水組Collagen I mRNA的表達(dá)高于碘伏＋雙氧水處理組（P＜0.05），正常小鼠則是碘伏+雙氧水組Collagen I mRNA表達(dá)高于生理鹽水和硼酸組。 結(jié)論： 1.不管在早期還是中晚期，硼酸處理對(duì)提高糖尿病小鼠傷口愈合率，促進(jìn)創(chuàng)面愈合均達(dá)到了很好的效果； 2.硼酸促進(jìn)糖尿病小鼠創(chuàng)面愈合的作用機(jī)制可能與其特殊的酸性條件能更好地刺激創(chuàng)面Tgf-β1、Collagen I mRNAs的表達(dá)有關(guān)； 3.正常小鼠早期使用硼酸和生理鹽水都能較好地促進(jìn)創(chuàng)面愈合，晚期使用碘伏+雙氧水對(duì)促進(jìn)創(chuàng)面愈合效果更好。
[Abstract]:Background and purpose:
Diabetic refractory wounds include diabetic foot and other diabetic wounds. Prolonged wounds caused by various causes are one of the most serious and costly chronic complications of diabetes. Severe diabetic foot can lead to amputation or even death. The pathogenesis and nursing of diabetic refractory wounds is a difficult and hot topic in clinical research.
TGF-beta 1/Smads signal transduction pathway is one of the important signal transduction pathways in repairing diabetic refractory wounds. Proliferation, chemotaxis, and stimulation of fibroblasts to produce extracellular matrix. Studies have shown that the formation of refractory diabetic wounds is related to the absolute or relative inadequacy of TGF-beta 1 expression in the wounds. In recent years, a few clinical cases have reported that weak acid environment can promote the healing of refractory wounds. In addition to biological inhibition, it may also be related to its possible stimulation of Tgf-beta 1 mRNA expression in wounds, which may be one of the important mechanisms of promoting wound healing and worthy of further study.
4% boric acid solution as a common clinical disinfectant, with weak acidity and low cytotoxicity, is often used for disinfection of skin, mucosa and wounds. Some clinical studies have also used boric acid on lower extremity ulcer, bedsore and other wounds and achieved good results. The experimental study on the nursing effect and related mechanism of refractory wound healing is expected to provide a more scientific reference for the refinement and revision of nursing measures for refractory wounds of diabetes mellitus.
Materials and methods:
1. Animal grouping and model establishment: 30 8-week-old male C57BL/6 mice were purchased and fed in SPF class Animal Laboratory of Experimental Animal Center of Third Military Medical University. They were randomly divided into diabetes group and normal control group, 15 mice in each group. Induced modeling, the cornea ring drill made mouse back wound.
2. The wounds of mice in different groups were cleaned and dressed once a day with 0.9% normal saline (A), 4% boric acid (B) and 0.5% iodophor + 3% hydrogen peroxide (C), respectively. The scab formation, healing and wound size were observed and recorded.
3. Trizol was used to extract the total RNA from the wound tissue of mice at different time points. After detecting the concentration of RNA and RT-PCR reaction, the cDNA was obtained.
4. The relative expression of Tgf-beta 1, Smad4 and Collagen I in the above wound tissues was detected by SYBR Green Q-PCR.
Result:
Effect of 1. boric acid on wound healing rate
The wound healing rate of diabetic boric acid treatment group was higher than that of other treatment groups at each time point (P < 0.05).
Effect of 2. boric acid on the expression of Tgf- beta 1mRNA in the wound
The expression of Tgf-beta 1 mRNA in the boric acid treated group was significantly higher than that in the other groups at 3 days after injury (P < 0.05), and the expression of Tgf-beta 1 mRNA in the boric acid treated group was significantly higher than that in the other groups at 3 days and 14 days after injury (P < 0.01).
Effect of 3. boric acid on Smad4mRNA expression in wound surface
Smad4 mRNA expression in normal mice treated with boric acid at 14 days after injury was lower than that in other treatment groups (P < 0.05); Smad4 mRNA expression in normal saline group at 14 days after injury was lower than that in iodophor + hydrogen peroxide treatment group (P < 0.05).
Effect of 4. boric acid on the expression of Collagen I mRNA in wound
The expression of Collagen I mRNA in boric acid treatment group and iodophor + hydrogen peroxide treatment group was higher than that in normal saline group at 3 days after injury (P < 0.05); at 14 days, the expression of Collagen I mRNA in boric acid group and normal saline group was higher than that in iodophor + hydrogen peroxide treatment group (P < 0.05). The expression of Collagen I mRNA in iodophor + H2O2 group was higher than that in normal saline and boric acid group.
Conclusion:
1. Boric acid treatment can improve the wound healing rate and promote the wound healing of diabetic mice in the early and middle and late stages.
2. The mechanism of the effect of boric acid on wound healing in diabetic mice may be related to the special acidic condition which can better stimulate the expression of Tgf-beta 1 and Collagen I mRNAs.
3. Early use of boric acid and normal saline can promote wound healing in normal mice, and late use of iodophor and hydrogen peroxide can promote wound healing better.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R473.5

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