微囊化基因修饰细胞复合组织工程真皮构建及其对创面愈合的影响

EFFECT OF BIOENGINEERED DERMIS INCORPORATED WITH MICROENCAPSULATED GENE MODIFIED CELLS ON WOUND HEALING

目的探讨微囊化神经生长因子(nerve grow th factor,NGF)基因修饰的N IH 3T 3细胞复合组织工程真皮的可行性及其对急性创面愈合的影响。方法构建分泌型NGF真核表达质粒pcDNA 3.1+/NGF并导入N IH 3T 3细胞,筛选出稳定表达的细胞株,将此细胞包入海藻酸钠-多聚赖氨酸-海藻酸钠微囊中培养。以Ⅰ、Ⅲ型胶原为基质、人成纤维细胞和制备好的微囊为种子细胞,用组织工程皮肤培养系统构建复合微囊的组织工程真皮,体外培养1周后行羟脯氨酸(hydroxypro line,Hyp)测定,EL ISA法测定其分泌功能,组织形态学观察。以猪背部急性全层创面为模型,按移植物不同分为5组:NGF-N IH 3T 3微囊真皮组(A组)、N IH 3T 3微囊真皮组(B组)、空囊真皮组(C组)、NGF(5 ng/m l)+胶原膜组(D组)和空白对照组(E组),观察创面再上皮化及愈合速度和愈合时间。结果转NGF基因微囊复合组织工程真皮后体外培养6周,仍稳定分泌NGF(124.32 pg/m l),体外培养1周后,组织工程真皮中NGF-N IH 3T 3微囊组Hyp含量为69.68±6.20 m g/g,较空白对照组增加约2倍以上。移植治疗急性创面,A组平均愈合时间为25±2 d,B组为34±3 d,C组为34±2 d,D组为33±2 d,E组为40±3 d,A组创面愈合时间平均至少缩短约10 d,提高了创面愈合的速度。结论将分泌NGF微囊复合组织工程真皮后可提高组织工程真皮的质量,并促进急性创面的愈合。

Objective To construct a bio-engineered dermis containing microencapsulated nerve growth factor （NGF） expressing NIH3T3 cells and to study the effect of the microencapsule on the bio-engineered dermis and acute wound healing. Methods A recombinant NGF （PcDNA3. I＋/NGF） was constructed and transfected into NIH-3T3 cells using FuFENETM6 transfection reagent. Positive cell strain was cultured and enclosed in alginate polylysine- alginate（APA） microcapsu[es in vitro. Bio-engineered dermis was incorporated with NGF-expressing micorencapsules and human fibroblast cells as seed cells using tissue engineering method. The characteristics of the dermis were described by the content of Hydroxyproline （Hyp）, HE staining. The content of NGF in the dermis culturing supernatant was measured by ELISA method. These bioengineered dermis were transplanted onto the acute circular full thickness excisional wounds on the dorsum of each swine to observe the rate of re-epithelization and wound healing： NGF-NIH3T3 microencapsulations （group A）, NIH3T3 microencapsulations （ group B）, empty microencapsulations （group C）, NGF incorporated with collagen Ⅰ （ group D） and blank （group E as control group）. Results NGF can be tested stably about 124.32 pg/ml in the dermis culturing supernatant after 6 weeks, and the content of Hyp in group A was 69.68±6.20（mg/g wet weight） and increased about 2 times when compared with control groups after 1 week. The tissue engineering skin grafts which can secrete NGF were used to cure the acute wounds and the rate of re-epithelization was promoted. The periods of wound healing were 25±2 days in group A, 34±3 days in group B, 34±2 days in group C, 33±2 days in group D and 40±3 days in group E. The period of wound healing was decreased about 10 days at least.Conclusion NGF-expressing NIH3T3 microencapsulates can promote the quality of bioengineered dermis and also promote acute wound healing.