Hsp90α在小鼠烫伤创面的作用研究

发布时间：2018-03-10 03:34

本文选题：深Ⅱ度烧伤　切入点：热休克蛋白　出处：《第四军医大学》2013年硕士论文　论文类型：学位论文

【摘要】：严重烧伤创面是各种皮肤创面中较难愈合的一种。烧伤病人在恢复过程中要面临诸多问题，但最为重要的还是加速愈合及减轻愈合后的瘢痕。因此，对于创面的处理，治疗以及如何缩短愈合时间成为临床工作者和研究学者所面临的主要挑战。愈合，即重塑受损组织的完整性。通过机体自身的机能来代替和修复受损部位，也可通过施加外在干预加速自然愈合过程。外在干预包括表面用药，敷料及手术等。而表面药物中，生长因子PDGF-BB被公认为能有效的加速创面愈合，但由于其高额的费用和致癌的潜能使其在临床上应用受限。此外，皮肤中广泛存在的TGF-β3同时也抑制了生长因子的促创面愈合效能。因此，究竟何物质才能真正有效的促进创面愈合成为目前研究的一个新重点。最新研究表明，，hsp90α在创面愈合过程中起到十分重要的作用。Hsp90α分子伴侣，是热休克蛋白家族中的一员，广泛存在于哺乳动物细胞体内，参与维持细胞的稳态。当细胞受到热力，缺氧，辐射等外在刺激后，可分泌到胞外参与组织修复。与生长因子相比，其不仅能够促进表皮细胞的迁移，还能够促进真皮成纤维细胞向创面迁移，并且不受TGF-β3的抑制作用，而且高糖环境仍不影响其促愈合效果。因此，hsp90α成为创面愈合研究的新热点。然而其在烧伤这一特殊创面，发挥何种效果，是否也能通过促进细胞迁移和增殖，加速烧伤创面修复再生，值得我们探讨。前期试验研究发现，烫伤后创面hsp90α RNA水平一过性表达增高，蛋白水平也相应增高，证实热应激后该蛋白表达有所变化，可能在促烫伤创面愈合中发挥作用。体外实验通过划痕和流式技术，进一步证实其促细胞迁移及增殖作用。最后通过在体实验大体观上验证其促愈合效果。明确hsp90α蛋白剂量与烧伤创面愈合相关，为烧伤创面的愈合研究提供一个新的思路。 1.目的：通过对深Ⅱ度烫伤小鼠皮肤创面hsp90α表达变化的研究，探讨其在烧伤这一特殊创面，发挥何种效果，是否也能通过促进细胞迁移和增殖，加速烧伤创面的修复再生。 2.主要方法： 2.1.小鼠深Ⅱ度烫伤模型的建立。雄性巴比赛（BALB/c）小鼠85只（20±1g），按随机数字表法将小鼠分为5组，正常对照组5只，烫伤2、4、6、8s每组各20只。烫伤前24h用1%戊巴比妥钠腹腔注射麻醉，10%硫化钠背部脱毛。自制烫伤仪（蒸汽温度92℃，直径2cm）在小鼠背部脱毛区域烫2、4、6、8s。烫伤后即刻、12、24、48h四个时间点切取皮肤样本（每一时相点标本数为5个），4%多聚甲醛固定，苏木精-伊红染色法（HE染色）检测烫伤深度，观察其致伤后皮肤烫伤深度的发展变化。 2.2.另取雄性BALB/c小鼠25只（20±1g），深Ⅱ度烫伤后，按随机数字表法将小鼠分为2组，免疫组化组（n=5）和经皮样分压组（n=20），分别用免疫组化法和经皮氧分压法检测该模型的缺氧情况，实验数据进行单因素方差分析。 2.3.雄性BALB/c小鼠55只（20±1g），深Ⅱ度烫伤后，按随机数字表法将小鼠分为3组，免疫组化组（n=10），Real-time PCR组(n=24)和Western-blot组（n=21）。分别用免疫组化法，Real-time PCR法和Western-blot法检测创面hsp90α的表达变化。免疫组化组取材为：正常组织/烫伤组织=1/1，时间点为烫后12h和48h。Real-time PCR组取材部位为创缘，时间点为烫后0、0.5、1、3、6、12及24h。Western-blot组取材部位为创缘，时间点为烫后6、12、24、48、72h及7d。 2.4.培养人表皮细胞系，建立细胞的热休克模型。将细胞接种于6孔板内，铺满后封口膜封住六孔板周围缝隙，将该板置于45℃水浴锅中15min。按热休克后0、0.5、1、3、6、12及24h这几个时间点收取细胞，检测细胞hsp90αRNA表达变化。同时，将细胞分为三组：热休克组，热休克+hsp90α组，热休克+17-DMAG组。检测不同处理组细胞迁移能力及凋亡和周期的变化。 2.5.雄性BALB/c小鼠30只（20±1g），深Ⅱ度烫伤后，按随机数字表法将小鼠分为3组：烫伤组，烫伤+外用hsp90α组，腹腔注射17-DMAG烫伤组。每天同一时刻给予处理，连续处理5天，观察创面愈合情况，每组在第七天时取该组的一半小鼠处死，切取带有正常皮肤的长条状创面，做HE染色，观察上皮化情况。 3.主要实验结果： 3.1.烫伤深度与烫伤时间密切相关，烫伤4s后，皮肤表皮全层及真皮深层损伤，皮肤附件-毛囊结构健存，即深Ⅱ度烫伤。且烫伤深度随着时间逐步发展，在烫伤后24h不再加深，达到稳定。其病理结果在24h后趋于一致。 3.2.烫伤创面为缺氧环境，深Ⅱ度烫伤72h后，小鼠背部皮下氧分压为37.0±2.7mm/Hg，而正常小鼠背部皮下氧分压为53.1±2.4mm/Hg（F=100.531，p＜0.05）。免疫组织化学染色结果显示烫伤创缘部位缺氧最为明显。 3.3.深Ⅱ度烫伤后，皮肤hsp90α表达一过性增高。免疫组织化学结果显示烫伤后12h组织hsp90α染色呈强阳性。Real-time PCR结果显示皮肤hsp90α RNA水平在烫后6h达高峰，为正常对照组的20倍以上。其蛋白水平也相应增高，时间滞后于RNA水平，于48h达高峰，随即逐渐下降。 3.4.表皮细胞热休克后，hsp90α RNA水平一过性增高。0.5h呈高峰。划痕实验发现，与单纯热休克组相比，热休克细胞加入hsp90α能够能够明显加快细胞的迁移速率，而加入hsp90α的抑制剂17-DMAG，则延缓了细胞的迁移速率（p 0.05）。同时，hsp90α能够促进细胞增殖，抑制细胞凋亡，加入17-DMAG则加速了细胞凋亡（p 0.05）。 3.5.与单纯烫伤组相比，外用hsp90α可明显加速深Ⅱ度烫伤创面的愈合，加快表皮爬行速度。腹腔注射17-DMAG，则延缓了创面的愈合。 4.结论： 4.1.雄性BALB/c小鼠（20±1g），蒸汽致伤4s,24h后取材，可建立稳定的小鼠深Ⅱ度烫伤模型。 4.2.激光多普勒经皮氧分压及免疫组织化学染色结果示烫伤部位为缺氧环境，在创缘部位缺氧最为明显。 4.3.与正常组小鼠相比，烫伤后小鼠皮肤hsp90α RNA及蛋白水平均一过性表达增高。 4.4.外用hsp90α对热休克表皮细胞有促迁移作用，该作用可被17-DMAG有效抑制。并且hsp90α能够通过促进细胞增殖，抑制细胞凋亡，加速烫伤创面愈合。 4.5.给予深Ⅱ度烫伤小鼠外用hsp90α治疗，可加速创面愈合，该蛋白可能是促进烧伤创面愈合的关键因子。
[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.

【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R644

【引证文献】