本文選題：深Ⅱ度燒傷　切入點(diǎn)：熱休克蛋白　出處：《第四軍醫(yī)大學(xué)》2013年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：嚴(yán)重?zé)齻麆?chuàng)面是各種皮膚創(chuàng)面中較難愈合的一種。燒傷病人在恢復(fù)過(guò)程中要面臨諸多問(wèn)題，但最為重要的還是加速愈合及減輕愈合后的瘢痕。因此，對(duì)于創(chuàng)面的處理，治療以及如何縮短愈合時(shí)間成為臨床工作者和研究學(xué)者所面臨的主要挑戰(zhàn)。愈合，即重塑受損組織的完整性。通過(guò)機(jī)體自身的機(jī)能來(lái)代替和修復(fù)受損部位，也可通過(guò)施加外在干預(yù)加速自然愈合過(guò)程。外在干預(yù)包括表面用藥，敷料及手術(shù)等。而表面藥物中，生長(zhǎng)因子PDGF-BB被公認(rèn)為能有效的加速創(chuàng)面愈合，但由于其高額的費(fèi)用和致癌的潛能使其在臨床上應(yīng)用受限。此外，皮膚中廣泛存在的TGF-β3同時(shí)也抑制了生長(zhǎng)因子的促創(chuàng)面愈合效能。因此，究竟何物質(zhì)才能真正有效的促進(jìn)創(chuàng)面愈合成為目前研究的一個(gè)新重點(diǎn)。 最新研究表明，，hsp90α在創(chuàng)面愈合過(guò)程中起到十分重要的作用。Hsp90α分子伴侶，是熱休克蛋白家族中的一員，廣泛存在于哺乳動(dòng)物細(xì)胞體內(nèi)，參與維持細(xì)胞的穩(wěn)態(tài)。當(dāng)細(xì)胞受到熱力，缺氧，輻射等外在刺激后，可分泌到胞外參與組織修復(fù)。與生長(zhǎng)因子相比，其不僅能夠促進(jìn)表皮細(xì)胞的遷移，還能夠促進(jìn)真皮成纖維細(xì)胞向創(chuàng)面遷移，并且不受TGF-β3的抑制作用，而且高糖環(huán)境仍不影響其促愈合效果。因此，hsp90α成為創(chuàng)面愈合研究的新熱點(diǎn)。然而其在燒傷這一特殊創(chuàng)面，發(fā)揮何種效果，是否也能通過(guò)促進(jìn)細(xì)胞遷移和增殖，加速燒傷創(chuàng)面修復(fù)再生，值得我們探討。 前期試驗(yàn)研究發(fā)現(xiàn)，燙傷后創(chuàng)面hsp90α RNA水平一過(guò)性表達(dá)增高，蛋白水平也相應(yīng)增高，證實(shí)熱應(yīng)激后該蛋白表達(dá)有所變化，可能在促燙傷創(chuàng)面愈合中發(fā)揮作用。體外實(shí)驗(yàn)通過(guò)劃痕和流式技術(shù)，進(jìn)一步證實(shí)其促細(xì)胞遷移及增殖作用。最后通過(guò)在體實(shí)驗(yàn)大體觀上驗(yàn)證其促愈合效果。明確hsp90α蛋白劑量與燒傷創(chuàng)面愈合相關(guān)，為燒傷創(chuàng)面的愈合研究提供一個(gè)新的思路。 1.目的： 通過(guò)對(duì)深Ⅱ度燙傷小鼠皮膚創(chuàng)面hsp90α表達(dá)變化的研究，探討其在燒傷這一特殊創(chuàng)面，發(fā)揮何種效果，是否也能通過(guò)促進(jìn)細(xì)胞遷移和增殖，加速燒傷創(chuàng)面的修復(fù)再生。 2.主要方法： 2.1.小鼠深Ⅱ度燙傷模型的建立。雄性巴比賽（BALB/c）小鼠85只（20±1g），按隨機(jī)數(shù)字表法將小鼠分為5組，正常對(duì)照組5只，燙傷2、4、6、8s每組各20只。燙傷前24h用1%戊巴比妥鈉腹腔注射麻醉，10%硫化鈉背部脫毛。自制燙傷儀（蒸汽溫度92℃，直徑2cm）在小鼠背部脫毛區(qū)域燙2、4、6、8s。燙傷后即刻、12、24、48h四個(gè)時(shí)間點(diǎn)切取皮膚樣本（每一時(shí)相點(diǎn)標(biāo)本數(shù)為5個(gè)），4%多聚甲醛固定，蘇木精-伊紅染色法（HE染色）檢測(cè)燙傷深度，觀察其致傷后皮膚燙傷深度的發(fā)展變化。 2.2.另取雄性BALB/c小鼠25只（20±1g），深Ⅱ度燙傷后，按隨機(jī)數(shù)字表法將小鼠分為2組，免疫組化組（n=5）和經(jīng)皮樣分壓組（n=20），分別用免疫組化法和經(jīng)皮氧分壓法檢測(cè)該模型的缺氧情況，實(shí)驗(yàn)數(shù)據(jù)進(jìn)行單因素方差分析。 2.3.雄性BALB/c小鼠55只（20±1g），深Ⅱ度燙傷后，按隨機(jī)數(shù)字表法將小鼠分為3組，免疫組化組（n=10），Real-time PCR組(n=24)和Western-blot組（n=21）。分別用免疫組化法，Real-time PCR法和Western-blot法檢測(cè)創(chuàng)面hsp90α的表達(dá)變化。免疫組化組取材為：正常組織/燙傷組織=1/1，時(shí)間點(diǎn)為燙后12h和48h。Real-time PCR組取材部位為創(chuàng)緣，時(shí)間點(diǎn)為燙后0、0.5、1、3、6、12及24h。Western-blot組取材部位為創(chuàng)緣，時(shí)間點(diǎn)為燙后6、12、24、48、72h及7d。 2.4.培養(yǎng)人表皮細(xì)胞系，建立細(xì)胞的熱休克模型。將細(xì)胞接種于6孔板內(nèi)，鋪滿后封口膜封住六孔板周?chē)p隙，將該板置于45℃水浴鍋中15min。按熱休克后0、0.5、1、3、6、12及24h這幾個(gè)時(shí)間點(diǎn)收取細(xì)胞，檢測(cè)細(xì)胞hsp90αRNA表達(dá)變化。同時(shí)，將細(xì)胞分為三組：熱休克組，熱休克+hsp90α組，熱休克+17-DMAG組。檢測(cè)不同處理組細(xì)胞遷移能力及凋亡和周期的變化。 2.5.雄性BALB/c小鼠30只（20±1g），深Ⅱ度燙傷后，按隨機(jī)數(shù)字表法將小鼠分為3組：燙傷組，燙傷+外用hsp90α組，腹腔注射17-DMAG燙傷組。每天同一時(shí)刻給予處理，連續(xù)處理5天，觀察創(chuàng)面愈合情況，每組在第七天時(shí)取該組的一半小鼠處死，切取帶有正常皮膚的長(zhǎng)條狀創(chuàng)面，做HE染色，觀察上皮化情況。 3.主要實(shí)驗(yàn)結(jié)果： 3.1.燙傷深度與燙傷時(shí)間密切相關(guān)，燙傷4s后，皮膚表皮全層及真皮深層損傷，皮膚附件-毛囊結(jié)構(gòu)健存，即深Ⅱ度燙傷。且燙傷深度隨著時(shí)間逐步發(fā)展，在燙傷后24h不再加深，達(dá)到穩(wěn)定。其病理結(jié)果在24h后趨于一致。 3.2.燙傷創(chuàng)面為缺氧環(huán)境，深Ⅱ度燙傷72h后，小鼠背部皮下氧分壓為37.0±2.7mm/Hg，而正常小鼠背部皮下氧分壓為53.1±2.4mm/Hg（F=100.531，p＜0.05）。免疫組織化學(xué)染色結(jié)果顯示燙傷創(chuàng)緣部位缺氧最為明顯。 3.3.深Ⅱ度燙傷后，皮膚hsp90α表達(dá)一過(guò)性增高。免疫組織化學(xué)結(jié)果顯示燙傷后12h組織hsp90α染色呈強(qiáng)陽(yáng)性。Real-time PCR結(jié)果顯示皮膚hsp90α RNA水平在燙后6h達(dá)高峰，為正常對(duì)照組的20倍以上。其蛋白水平也相應(yīng)增高，時(shí)間滯后于RNA水平，于48h達(dá)高峰，隨即逐漸下降。 3.4.表皮細(xì)胞熱休克后，hsp90α RNA水平一過(guò)性增高。0.5h呈高峰。劃痕實(shí)驗(yàn)發(fā)現(xiàn)，與單純熱休克組相比，熱休克細(xì)胞加入hsp90α能夠能夠明顯加快細(xì)胞的遷移速率，而加入hsp90α的抑制劑17-DMAG，則延緩了細(xì)胞的遷移速率（p 0.05）。同時(shí)，hsp90α能夠促進(jìn)細(xì)胞增殖，抑制細(xì)胞凋亡，加入17-DMAG則加速了細(xì)胞凋亡（p 0.05）。 3.5.與單純燙傷組相比，外用hsp90α可明顯加速深Ⅱ度燙傷創(chuàng)面的愈合，加快表皮爬行速度。腹腔注射17-DMAG，則延緩了創(chuàng)面的愈合。 4.結(jié)論： 4.1.雄性BALB/c小鼠（20±1g），蒸汽致傷4s,24h后取材，可建立穩(wěn)定的小鼠深Ⅱ度燙傷模型。 4.2.激光多普勒經(jīng)皮氧分壓及免疫組織化學(xué)染色結(jié)果示燙傷部位為缺氧環(huán)境，在創(chuàng)緣部位缺氧最為明顯。 4.3.與正常組小鼠相比，燙傷后小鼠皮膚hsp90α RNA及蛋白水平均一過(guò)性表達(dá)增高。 4.4.外用hsp90α對(duì)熱休克表皮細(xì)胞有促遷移作用，該作用可被17-DMAG有效抑制。并且hsp90α能夠通過(guò)促進(jìn)細(xì)胞增殖，抑制細(xì)胞凋亡，加速燙傷創(chuàng)面愈合。 4.5.給予深Ⅱ度燙傷小鼠外用hsp90α治療，可加速創(chuàng)面愈合，該蛋白可能是促進(jìn)燒傷創(chuàng)面愈合的關(guān)鍵因子。
[Abstract]:Severe burn is one of the more difficult to heal all kinds of skin wound in burn patients. Face many problems during the recovery process, but the most important thing is to accelerate healing and reduce scar after healing. Therefore, for the treatment of wound treatment, and how to shorten the healing time of a main challenge for clinicians and research scholars. Healing, namely remodeling of damaged tissue integrity. Through the body's own function of the bodyitself, also available through external intervention to accelerate the natural healing process. The external intervention including surface treatment, dressing and surgery. While the surface of drugs, PDGF-BB is recognized as a growth factor can effectively promote wound healing but, because of its high cost and carcinogenic potential of the clinical application is limited. In addition, widely exists in the skin of TGF- beta 3 also inhibited growth factor promoting wound surface Healing efficiency is a new focus of research at the present time.
The latest research shows that Hsp90 plays a role of alpha.Hsp90 alpha molecular chaperone is very important in the process of wound healing, is a member of the heat shock protein family, widely exist in mammalian cells in vivo, involved in maintaining cellular homeostasis. When cells were heat, hypoxia, radiation and other external stimulation, can be secreted into the tissue repair participate in extracellular. Compared with the growth factor, which can not only promote the migration of epidermal cells, can also promote dermal fibroblasts to the wound migration, and is not affected by the inhibition of TGF- beta 3, and high glucose environment is still not affect the healing effect. Therefore, Hsp90 has become a new research hotspot of wound healing. However in this special burn wounds, play what effect, whether can promote cell migration and proliferation, accelerate wound repair and regeneration, is worth discussing.
The study found that after scald wound Hsp90 alpha RNA levels had a higher expression of the protein level increased, confirmed that the protein expression change after heat stress, may play a role in promoting wound healing. In vitro by scratch assay and flow cytometry further confirmed its role in promoting cell migration and proliferation at the end of the experiment in vivo. In view of the healing effect. To verify the clear Hsp90 protein dose and burn wound healing, provide a new idea for the study of wound healing.
1. purpose:
Through studying the change of Hsp90 alpha expression in skin wound of deep second degree scald mice, we can explore its effect in the special wound of burn, and whether it can also accelerate cell repair and regeneration by promoting cell migration and proliferation.
2. main methods:
The establishment of 2.1. mouse scald model. Male Pakistan contest (BALB/c) 85 mice (20 + 1g), were randomly divided into 5 groups of mice, 5 rats in normal control group, 20 rats in each group. 2,4,6,8s burn scald 24h with 1% sodium pentobarbital intraperitoneal injection anesthesia, 10% sodium sulfide back hair removal. Self-made wound meter (steam temperature of 92 DEG C, diameter 2cm) immediately in the back of the mice hair removal area 2,4,6,8s. after scalding hot, 12,24,48h four time points cut skin samples (each time point were number 5), 4% paraformaldehyde, hematoxylin eosin staining (HE staining) detection the burn depth, observe the change of development after injury in scalded skin depth.
2.2. another 25 male BALB/c mice (20 + 1g), scald, were randomly divided into 2 groups of mice, immunohistochemistry group (n=5) and the partial pressure of dermoid (n=20) group, respectively with immunohistochemical method and transcutaneous oxygen pressure hypoxia detection of the model, data were analyzed by one-way ANOVA.
2.3. of 55 male BALB/c mice (20 + 1g), scald, were randomly divided into 3 groups of mice, immunohistochemistry group (n=10), Real-time PCR group (n=24) and Western-blot group (n=21) respectively. By immunohistochemical method, the expression change of Real-time and PCR Western-blot was used to measure the wound Hsp90 alpha. Immunohistochemistry group were: normal tissue / scald tissue =1/1, time point after 12h and 48h.Real-time hot PCR group based on the site for a margin, time point after the hot 0,0.5,1,3,6,12 and 24h.Western-blot group based on the site for a margin, time point after the hot 6,12,24,48,72h and 7d.
2.4. cultured human epidermal cells, the establishment of heat shock model cells. The cells were seeded in 6 well plates, after covered with a sealing film for sealing plate gap around the 15min. plate is arranged in a water bath at 45 DEG C after heat shock, 0,0.5,1,3,6,12 and 24h this time a few charge cells to detect the expression changes of Hsp90 alpha RNA cells at the same time, the cells were divided into three groups: heat shock group, heat shock +hsp90 Alpha Group, heat shock +17-DMAG group. Different treatment groups to detect the changes of cell migration and apoptosis and cycle.
2.5. of 30 male BALB/c mice (20 + 1g), scald, randomly were divided into 3 groups: scald group, scald + topical Hsp90 Alpha Group, intraperitoneal injection of 17-DMAG in scald group. The same time every day to give treatment, treatment for 5 days, to observe the wound healing. Half of the mice in each group on the seventh day of the group, cut the strip wound with normal skin, HE staining, observe the epithelium.
3. the main experimental results are as follows:
3.1. burn depth and scalding time is closely related to 4S after scald, the skin epidermis and dermis damage, skin appendages - healthy hair follicle structure, namely deep second degree burn and scald depth. With the gradual development of time in 24h after scald not deepened, is stable. The pathological results are consistent in 24h.
3.2. scalded wound is anoxic environment. After deep second degree scald 72h, the subcutaneous oxygen partial pressure of mice is 37 + 2.7mm/Hg, while the subcutaneous oxygen partial pressure of normal mice is 53.1 + 2.4mm/Hg (F=100.531, P < 0.05). Immunohistochemical staining shows that the location of burn wound margin is most obvious.
3.3. deep second degree scalded skin Hsp90 expression transiently. Immunohistochemistry showed 12h after scald tissue Hsp90 alpha positive staining results showed that.Real-time PCR Hsp90 alpha RNA levels in the skin after the hot 6h reached a peak of 20 times more than the normal control group. The protein level increased time behind the level of RNA, peaked at 48h, then decreased gradually.
3.4. epidermal cells after heat shock, Hsp90 RNA had a higher level of alpha.0.5h showed the peak. The scratch test found that, compared with the simple heat shock group, heat shock cells with Hsp90 alpha could significantly accelerate the migration rate of cells, while adding Hsp90 alpha inhibitor 17-DMAG, delayed cell migration rate (P 0.05) at the same time, Hsp90 was able to promote cell proliferation, inhibit apoptosis, joining 17-DMAG will accelerate the cell apoptosis (P 0.05).
Compared with the scald group, 3.5. Hsp90 can accelerate the healing of deep second degree scald wounds and accelerate the skin crawling speed. 17-DMAG intraperitoneal injection delayed the healing of wounds.
4. conclusion:
4.1. male BALB/c mice (20 + 1g), steam injuring 4S and 24h after 24h, can establish a stable model of deep second degree scald in mice.
The results of 4.2. laser Doppler's percutaneous oxygen partial pressure and immunohistochemical staining showed that the site of scald was anoxic environment, and the most obvious anoxia at the wound site.
Compared with the normal group, the level of Hsp90 alpha RNA and protein in the skin of the scalded mice was higher than that of the normal group.
4.4. external Hsp90 alpha can promote the migration of heat shock epidermal cells, which can be effectively inhibited by 17-DMAG. Hsp90 alpha can accelerate cell proliferation and inhibit cell apoptosis and accelerate wound healing.
4.5. can be used for the treatment of deep second degree scald mice with Hsp90 alpha, which can accelerate the healing of the wound. This protein may be the key factor to promote the healing of burn wounds.

【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類(lèi)號(hào)】：R644

【引證文獻(xiàn)】

相關(guān)期刊論文 前1條

1 劉宇;劉海源;陳穎;占潔;龔洪翰;;1.5T MRI靜磁場(chǎng)對(duì)小鼠燙傷創(chuàng)面修復(fù)作用初探[J];中國(guó)燒傷創(chuàng)瘍雜志;2015年05期

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