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目的观察与人类巨细胞病毒载体结合的转化生长因子β_1(PCMV_4-hTGFβ_1)重组质粒裸 DNA 直接注射于深Ⅱ度烫伤大鼠体内后的基因表达规律及对创面愈合的作用,探讨其作为核酸疫苗的可行性,为烧伤的基因治疗提供理论基础和实验依据。方法大鼠随机分为烫伤+核酸疫苗组;单纯核酸疫苗组;对照组。采用 ELISA 方法检测裸 DNA 注射后血清中中和抗体水平;Southern 杂交、原位杂交检测裸 DNA 注射后在肌肉中存留时间,组织特染法检测组织中Ⅰ/Ⅲ型胶原的变化。结果裸 DNA 肌注7天后血清中可检测出抗 TGFβ_1中和抗体,21天达其峰值,28天有轻度下降。Southern 杂交显示裸 DNA 注射后,5min 即可检测出高浓度 TGFβ_1,1h 后浓度开始下降,3h 后仍可检测出。原位杂交在肌注5天后,肌肉中还有阳性反应。核酸疫苗组创面愈合速度在前9天较对照组快,差异有显著性意义(P<0.05),在后期与对照组间差异无显著性意义(P>0.05),但Ⅰ/Ⅲ型胶原比例明显下降(P<0.01)。结论 PCMV_4-hTGFβ_1即可加速创面愈合,又可控制Ⅰ型胶原合成,控制瘢痕增生。证实了其作为核酸疫苗治疗烧伤创面及控制瘢痕形成是可行的。
OBJECTIVE: To observe the gene expression pattern of PCMV_4-hTGFβ_1 recombinant plasmid exposed to human cytomegalovirus vector and its effect on wound healing by direct injection into deep second degree scalded rats, The feasibility of providing a theoretical basis and experimental basis for the gene therapy of burns. Methods The rats were randomly divided into scald + nucleic acid vaccine group; pure nucleic acid vaccine group; control group. The level of neutralizing antibody in the serum was detected by ELISA. The hybrid tissues were stained by Southern hybridization and in situ hybridization. The retention time in the muscle after the naked DNA injection was detected. The type Ⅰ / Ⅲ collagen in the tissue was detected by TUNEL. Results Anti-TGFβ 1 neutralizing antibody was detected in the serum of nude mice after 7 days of intramuscular injection. The neutralizing antibody reached its peak on the 21st day and slightly decreased on the 28th day. Southern hybridization showed that high concentrations of TGFβ_1 could be detected after 5 min of naked DNA injection, and the concentration of TGFβ began to decrease after 1 h, and was still detectable after 3 h. In situ hybridization 5 days after intramuscular injection, there is a positive muscle reaction. The wound healing rate of nucleic acid vaccine group was faster than that of the control group in the first 9 days (P <0.05), but there was no significant difference between the latter group and the control group (P> 0.05) Significantly decreased (P <0.01). Conclusions PCMV_4-hTGFβ_1 can accelerate wound healing and control type Ⅰ collagen synthesis and control scar hyperplasia. Confirmed its feasibility as a nucleic acid vaccine for the treatment of burn wounds and the control of scar formation.