第三方未成熟树突状细胞负载异体抗原诱导免疫耐受的实验研究

目的观察第三方未成熟树突状细胞（imDC）负载同种异体抗原后对其免疫特性的影响。方法从健康足月新生儿脐血中分离单核细胞，采用重组人粒细胞巨噬细胞集落刺激因子（rhGM-SCg）和重组人白细胞介素（rhIL）4联合培养7d，诱导其分化成imDC，并通过光学显微镜和扫描电镜观察细胞形态、检测细胞表型及混合淋巴细胞反应（MLR）；将培养的细胞与异体淋巴细胞抗原共同孵育，并给予共刺激分子阻断剂细胞毒性T淋巴细胞相关抗原4免疫球蛋白（CTLA-4Ig）处理，检测抗原负载前后的细胞表型变化，通过MLR比较致敏前后T淋巴细胞增殖的能力。结果（1）培养后细胞具有典型的imDC特征，CD1α、CD83、CD80、CD86等成熟标志呈低表达，分别为21．42％、0．59％、5．39％、3．85％；人类白细胞DR抗原（HLA-DR）的表达率为60．66％，MLR结果提示其不能刺激同种异体T淋巴细胞增殖。（2）负载抗原后的DC表现出成熟特性，CD1α、CD83、CD80、CD86及HLA-DR表达明显增高，分别为65．51％、42．20％、56．45％、38．52％、76．44％（P〈0．05）；能够刺激T淋巴细胞增殖[刺激指数（SI）〉2．00]。（3）给予共刺激阻断剂CTLA-4Ig致耐处理后，SI由2．51下降到0．39。可明显抑制T淋巴细胞增殖。结论第三方imDC负载抗原后能表现出成熟特性；经CTLA-4Ig致耐处理，不能刺激未致敏T淋巴细胞增殖。有望诱导受体针对供者抗原的特异性免疫耐受。     

热休克预处理对肠上皮细胞缺氧-再给氧损伤的保护作用及其机制

目的 :观察热休克预处理对肠上皮细胞 (IEC 6 )缺氧再给氧损伤的影响 ,并探讨热休克蛋白 70(HSP70 )的细胞保护作用。方法 :体外培养 IEC 6细胞 ,分为正常对照组、单纯缺氧再给氧组及热休克预处理组 ,观察缺氧再给氧后各组细胞 HSP70及 Bcl 2表达及细胞活力、乳酸脱氢酶 (L DH)漏出情况 ;用流式细胞仪检测细胞凋亡情况。结果 :热休克预处理可诱导 HSP70产生 (P <0 .0 1) ,显著增加缺氧再给氧处理后IEC 6细胞活力 (P<0 .0 5 ) ,使 L DH漏出减少 (P<0 .0 5 ) ,凋亡相关基因 Bcl 2表达明显增加 (P<0 .0 1) ,细胞凋亡率明显降低 (P<0 .0 1)。结论 :热休克预处理可能通过诱导 HSP70表达来减轻肠上皮细胞缺氧再给氧损伤 ;增加细胞 Bcl 2基因表达并抑制肠上皮细胞缺氧再给氧后细胞凋亡可能是其保护作用机制之一。     

大鼠烫伤创面感染模型的研制

Objective To reproduce a reliable rat model of burn with infection for the study of prevention and treatment of infected wound. Methods ( 1 ) Electrical burn producing apparatus equipped with constant temperature (80 ℃ ) and pressure (0.5 kg) was used to reproduce burn injury (with area of 4.5 cm2 ) on both sides of the back in 50 SD rats for different duration (4, 6, 8, 10, 12 s) , with 10 rats for each burn duration. On post burn day (PBD) 1, gross condition of wounds was observed with naked eyes.Wounds on the left side were used to observe healing time. The wounds on the right side were used for histological observation to determine the depth of injury, and they were classified into superficial and deep partialthickness injury. (2) Another 36 SD rats were divided into A (inflicted with superficial partial-thickness burn, n = 18) and B (inflicted with deep partial-thickness burn, n = 18) groups according to the random number table. Rats in both groups were treated in accordance with method of preliminary experiment. Immediately after burn, 0. 1 mL of liquid containing 1 × 109, 1 × 107, 1 × 105 CFU Pseudomonas aeruginosa (PA) ATCC 27853 was respectively inoculated to the wounds on one side (with 6 rats for each amount) ,while the wounds on the other side were treated with the same volume of normal saline as control. Inflammatory reaction of wounds was examined with HE staining on post inoculation day (PID) 1. On PID 1, 2, 3,5, 7, and 14, the number of subeschar bacteria was respectively counted and the bacteria were identified with Gram stain and biochemical reaction. Wound healing time was recorded. Data were processed with t test. Results (1) Burn for 6, 8 s was respectively identified as injury time resulting in superficial or deep partial-thickness injury according to histological observation and wound healing time. (2) Obvious inflammatory cell infiltration was observed in the wounds in B group which were inoculated with 1 × 107 , 1 ×109 CFU PA, and the infiltration was less marked in A group with inoculation of 1 × 109 CFU PA. (3) The bacteria isolated from wounds of A and B groups was identified as PA. The subeschar bacteria count within PID 14 in A group, in which different amount of PA was inoculated, was mostly less than 1 × 105 CFU/g of tissue, while that in B group in which 1 × 109 CFU PA was inoculated was more than 1 × 105 CFU/g of tissue. (4) There was no obvious difference in wound healing time between wounds inoculated with different amount of PA and wounds treated with normal saline in A group ( with t value respectively 1.26, 0. 29, 1.07,P values all above 0.05 ). Wound healing time of wounds in B group, in which 1 × 109 CFU PA was inoculated, was longer as compared with that treated with normal saline [(22.5 + 1.0) d vs. ( 19.4 + 1.6) d, t =2.73, P ＜0. 05]. Conclusions In rat, deep partial-thickness burn wound inoculated with 1 × 109 CFU PA ATCC 27853 is a reliable model with high reproducibility for the study of infection of burn wound.     

HSP70对严重烧伤大鼠保护作用的实验研究

目的寻找对严重烧伤早期缺血缺氧性损伤的可行性保护途径.方法构建含HSP70的重组腺病毒载体,股静脉注射该腺病毒载体48 h后,检测大鼠严重烧伤后6 h其血清中内毒素及LDH、IL-1α、IL-1β、IL-6、TNF-α细胞因子水平.结果未转染组大鼠严重烧伤后6 h血清中内毒素及检测的细胞因子水平较正常对照组明显升高.静脉注射含HSP70重组腺病毒组大鼠严重烧伤后血清中上述指标的检测水平与未转染组比较明显降低,但仍显著高于正常对照组.结论静脉注射含HSP70重组腺病毒可减轻严重烧伤后大鼠的缺血缺氧性损伤,但不能完全阻断该损伤.     

高迁移率族蛋白B1cDNA的克隆与表达

目的克隆人高迁移率族蛋白B1（HMGB1）cDNA,构建重组原核表达载体,对其诱导表达并鉴定,为设计HMGB1 cDNA突变体及诱导表达可竞争性抑制HMGB1炎症效应的突变体蛋白奠定基础。方法提取人新鲜扁桃体组织总RNA,经RT-PCR扩增出人HMGB1 cDNA序列,并克隆至载体pUC18进行序列测定,随后构建于原核表达载体pQE-80L中,经IPTG诱导4小时后,可表达Mr约30 000的蛋白。Western Blotting鉴定所表达的目的蛋白。结果经RT—PCR扩增得到了648bp的cDNA,经序列分析与GenBank中报道的已知序列完全一致,构建了HMGB1蛋白的重组表达质粒,诱导表达了目的蛋白,经Western Blotting证实,在Mr约为30000处有一条清楚的蛋白带。结论获得了HMGB1 cDNA的克隆与原核表达载体。     

人高迁移率族蛋白的分子改建及原核表达

目的 构建人高迁移率族蛋白(high mobility group box 1 protein,HMGB1)突变体及表达与鉴定目的蛋白,为进一步通过HMGB1突变体蛋白与天然HMGB1竞争结合相应受体而抑制其致炎效应奠定了基础.方法 在已成功克隆和表达人HMGB1的基础上,采用一步反向PCR致突变法构建人HMGB1 cDNA的6个突变体,并分别克隆于原核表达载体pQE-80L上,表达6个相应的重组HMGB1突变体蛋白,并经Western blot鉴定.结果 获得人HMGB1 cDNA的6个突变体,并成功构建到原核表达载体pQE-80L上,诱导表达且经Western blot鉴定了目的蛋白.结论 成功构建人HMGB1突变体,并进行了目的蛋白表达的鉴定.     

HSP70基因静脉注射对严重烧伤大鼠肠粘膜保护作用的实验研究

目的 为严重烧伤后肠粘膜损伤的防治提供可行性的基因治疗途径。方法 大鼠静脉注射含HSP70基因的重组腺病毒载体，通过严重烧伤后肠粘膜病理形态学改变、损伤指数统计及血清中乳酸脱氢酶含量的变化观察基因转染后保护效应。结果 静脉转染HSP70基因的大鼠烧伤后肠粘膜组织病理改变较单纯烧伤组减轻，损伤指数明显降低，血清中乳酸脱氢酶含量明显下降。结论 大鼠静脉注射含HSP70基因的重组腺病毒载体可保护严重烧伤后肠粘膜的缺血缺氧性损伤。为严重烧伤早期缺血缺氧性损伤的防治提供可行性的基因治疗途径。     

三种抗生素治疗严重烧伤感染的效果对比分析

目的:比较头孢他啶、头孢哌酮-舒巴坦与亚胺培南-西拉司丁3种抗生素治疗严重烧伤感染的效果.方法:选择严重烧伤患者90例,随机分为头孢他啶、头孢哌酮-舒巴坦和亚胺培南-西拉司丁3组,每组各30例.头孢他啶和头孢哌酮-舒巴坦组均为每次1 g,静脉滴注,每日3次,疗程4～6天;亚胺培南-西拉司丁组每次0.5 g,静脉滴注,每日3次,疗程4～6天.所有患者创面均做分泌物细菌培养、菌种鉴定和药敏试验.结果:头孢他啶、头孢哌酮-舒巴坦及亚胺培南-西拉司丁组有效率分别为60.0%、73.3%及93.3%; 共培养细菌200株,其中以铜绿假单胞菌(30.5%)和金黄色葡萄球菌(22.0%)最为常见.所有革兰阴性杆菌对头孢他啶、头孢哌酮-舒巴坦和亚胺培南-西拉司丁的敏感率分别为69.0%、76.0%与94.0%,所有革兰阳性球菌对头孢他啶、头孢哌酮-舒巴坦和亚胺培南-西拉司丁的敏感率分别为68.0%、70.0%与94.0%.结论:严重烧伤感染患者选择亚胺培南-西拉司丁的治疗效果优于头孢他啶和头孢哌酮-舒巴坦,亚胺培南-西拉司丁可作为治疗严重烧伤感染的首选药物.     

互联网资源在烧伤外科医师继续教育的应用

我国住院医师和专科医师规范化培养制度才刚刚起步,在当前互联网高速发展的形势下,结合医学教育模式的变革,对如何充分利用互联网上丰富的资源,高效、便捷地对烧伤外科医师进行较高质量的继续教育进行了探讨.     

抗生素治疗导致细菌内毒素释放的研究进展

由革蓝阴性(G-)细菌引起的各种感染仍是目前临床上主要的并发症和死亡原因之一.即使在应用各种广谱、高效抗生素治疗感染取得巨大进步的今天,对抗菌治疗中产生的一系列严重反应,如发热、中毒性休克及多器官功能衰竭(MOF)等,仍然没有取得防治的根本性进展.其中抗生素治疗中导致G 细菌释放内毒素(lipoplysaccharide,LPS),以及由其引起的严重损害存在着十分复杂的作用机理没有完全阐明,是一个主要原因[1].