The Effect Of Fibroblasts Transfected By Transforming Growth Factor-β₃ On The Wound Healing Of Deep Secondary Rat Burn Model

Background and objective Wound healing is a very complicated process that involves withmolecular biology and cell biology. The wound repairs and heals by the homocellular orheterocellular regeneration of the injured region. Up till now, it is known that a number ofcytokines have participated in this process. Among them, TGF-βis one of the very importantones. Transforming growth factor-β(TGF-β) consists of a highly homologous family ofmultifunctional peptides which are differentially expressed and function in a wide range of targetcells. It mediates the migration and proliferation of fibroblast. It controls the synthesis anddegradation of collagenâ… ,â…¢, elastic fiber, fibronectin and extracellular matrix (ECM). In a word,it plays a very important role in the process of wound healing. There are three isoforms of TGF-βin mammalian. They are TGF-β₁,β₂ andβ₃. It is confirmed that TGF-β₃ could lower thelevel of TGF-β₁ andβ₂. What's more, it could accelerates the wound healing and reduces thescar formation. The transforming growth factor protein using locally in the wound would bequickly degradated by the proteinase in the wound effusion. Multiple or persistent administrationis needed to achieve certain effect. However, the transgenic technique is hopefully considered tobe a solution.Deep secondary rat burn model was adopted in this experiment. The PLASTN plasmidcontaining TGF-β₃ cDNA was constructed using reconstructed retrovirus as vehicle. Lipoplastmethod was applied to import the plasmid into the incasing cell PA317. And then the fibroblastswere transfected and transplanted onto the wound surface. At the mean time, SulfadiazinmuArgenteum (SD-Ag), Epidermal growth factor (EGF) and human reconstructed transforminggrowth factor (rhTGF-β₃) protein were used to treat the burnt rat. The change ofhistomorphology of the deep secondary burnt wound, the wound healing rate, the moisturecapacity of the wound, the expression of TGF-β₁,β₂ andβ₃ were observed after burnt. Thepossibility of continuingly using the deep secondary bum model as the transgenic animal modelof the next experiment research was evaluated. The effect of TGF-β₃ transgenic fibroblast onwound healing was approached and this effect would help as a guideline in future clinical application.Materials and method Ninety adult Wistar rats of clean level weighing 225±25g wereused in the experiment. The rats were randomly divided into three groups: the SD-Ag group,the epidermal growth factor (EGF) group, the fibroblasts transfected by transforming growthfactor-β₃ (TGF-β₃) group. After 24 hours of depilation, the rats were anesthetized, and then theexposed skin was submerged into the 80℃hot water for 15 seconds. Intraperitoneal injection of5ml Lactated Ringer's solution was adopted in order to antishock after that. The rats were bred indifferent cages. 1) The SD-Ag group: the wound area of each rat was marked in a translucentpaper 1, 3, 7, 14 and 21 days after burnt. Exogenous SD-Ag coverage was given after that. 2)The EGF group: besides the same procedures handled in the SD-Ag group, exogenous EGF(20μg/L) was applied onto the wound surface. 3) TGF-β₃ group: besides the same procedureshandled in the SD-Ag group, the transgenic TGF-β₃ fibroblasts (10⁵/ml) was applied to thewound surface. The rats were killed 1, 3, 7, 14 and 21 days after burnt. The whole thickness ofthe burnt skin was harvested. Part of the skin was fixed in 4% paraformaldehyde and embeddedin paraffin. A serial of slices of 2μm thickness were made and stained with hematoxylin andeosin. HE stain was used to observe the morphological changes in the wound tissue. Translucentpaper converting weigh method was adopted to calculate the wound healing rate. The dry wetweigh method was applied to compute the moisture capacity of the wound. S-P method was usedto observe the expression of TGF-β₁,β₂ andβ₃ in the burnt skin tissue. Image analysissoftware was employed to determine the mean optical density of TGF-β₁,β₂ andβ₃.Analysis of variance and correlation analysis were used to make a statistical analysis of theindexes mentioned above.Resultsâ‘ The animal condition and the observation of the wound surface: 90 rats all survivedbefore the execution. The wound surface of the SD-Ag group was a little bit engorged one or twodays after burnt. A little exudation and red swelling was seen. The inflammation was lighter inEGF group and lightest in TGF-β₃ group. The wound became dry in all three groups three daysafter burnt. Dry crust was formed respectively in succession. There was no abnormal secretionunder the crust. No adverse reaction was seen either.â‘¡The comparison of wound healing rate:the healing rate was EGF group＞TGF-β₃ group＞SD-Ag group 7 days after burnt. There wassignificant difference between EGF group and SD-Ag group (P＜0.05). But there was no significant difference between EGF group and TGF-β₃ group as well as TGF-β₃ group andSD-Ag group (P＞0.05); 14 days after burnt, the healing rate was TGF-β₃ group＞EGFgroup＞SD-Ag group. There was significant value between TGF-β₃ group and EGF group(P＜0.05), while remarkably difference was found between TGF-β₃ group and SD-Ag group(P＜0.01). There was no significant difference between EGF group and SD-Ag group(P＞0.05); 21 days after burnt, the healing rate was still TGF-β₃ group＞EGF group＞SD-Aggroup. The difference between TGF-β₃ group and EGF group was significant (P＜0.05) whilethe difference between TGF-β₃ group and SD-Ag group was dramatically significant (P＜0.01).There was significant value between EGF group and SD-Ag group (P＜0.05).â‘¢Thecomparison of moisture capacity in the wound surface: There was no significant differencebetween the three groups 1, 3 and 7 days after burnt (P＞0.05) ; 14 days after burnt, there was nosignificant difference between TGF-β₃ group and EGF group as well as EGF group and SD-Aggroup (P＞0.05). But the difference between TGF-β₃ group and SD-Ag group was significant(P＜0.05); 21 days after burnt, the difference between TGF-β₃ group and EGF group wassignificant (P＜0.05), and the difference between TGF-β₃ group and SD-Ag group wasremarkably significant (P＜0.01). But there was no significant value between EGF group andSD-Ag group (P＞0.05).â‘£The observation of pathohistology: A typical deep secondary burnwound was identified by tissue section one day after burnt. The epidermis, part of dermis and theskin appendages were obviously injured. Inflammatory cell infiltration was seen too. 3 and 7days after burnt, the inflammation and swelling became severe. There was immature fibroblastsproliferation in connective tissue. There was vascular proliferation in EGF group. Fibroblastproliferation was less in TGF-β₃ group. 14 days after burnt, there was massive proliferation offibroblasts in connective tissue. Epithelial proliferation was seen under the crust of EGF groupand TGF-β₃ group. 21 days after burnt, there was still a small scale of inflammatory cellinfiltration in the tissue. Most of the wound of TGF-β₃ group healed under the crust.⑤Theexpression of TGF-β₁ in skin tissue: there was no significant difference between the threegroups one and three days after burnt (P＞0.05); 7 days after bumt, the difference betweenSD-Ag group and TGF-β₃ group was significant (P＜0.05). A remarkably significant differencewas found between EGF group and TGF-β₃ group (P＜0.01) ; 14 days after burnt, there wassignificant difference between SD-Ag group and TGF-β₃ group as well as TGF-β₃ group and EGF group (P＜0.05); 21 days after burnt, there was remarkably significant difference betweenSD-Ag group and TGF-β₃ group as well as TGF-β₃ group and EGF group (P＜0.01). Thedifference between EGF group and SD-Ag group was not significant 7, 14 and 21 days afterburnt (P＞0.05).â‘¥The expression of TGF-β₂ in skin tissue: there was no significant valuebetween the three groups 1 and 3 days after burnt (P＞0.05); 7 and 14 days after burnt, there wassignificant difference between SD-Ag group and TGF-β₃ group as well as TGF-β₃ group andEGF group (P＜0.05). But there was no significant difference between EGF group and SD-Aggroup (P＞0.05); 21 days after burnt, There was remarkably significant difference betweenSD-Ag group and TGF-β₃ group as well as TGF-β₃ group and EGF group (P＜0.01). But therewas no significant difference between EGF group and SD-Ag group (P＞0.05).⑦Theexpression of TGF-β₂ in skin tissue: there was remarkably significant difference betweenSD-Ag group and TGF-β₃ group as well as TGF-β₃ group and EGF group 1, 3, 7, 14 and 21days after burnt (P＜0.01 ). But the difference was not significant between EGF group and SD-Aggroup (P＞0.05).⑧The results of correlation of analysis: the wound healing rate was positivelycorrelated to the MOD values of TGF-β₃ (r=0.348, p＜0.01); but it was negatively correlatedto the moisture capacity of the wound (r=-0.524, p＜0.01 ), the MOD values of TGF-β₁ (r=-0.984, p＜0.01 )and the MOD values of TGF-β₂ (r=-0.842, p＜0.01). The MOD valuesof TGF-β₁ was positively correlated to the MOD values of TGF-β₃ (r=0.662, p＜0.01), butwas negatively correlated to the MOD values of TGF-β₃(r=-0.434, p＜0.01).Conclusionâ‘ The deep secondary rat burn model of this experiment was easy to establish andduplicate. And it was identified to be deep secondary burn by pathologist. It could be repeatedeasily.â‘¡Wound healing rate was adopted to measure the wound area in this experiment. Itreflected the different healing conditions of the three groups in different time after burnt. And itwas correlated to the expression of TGF-β₁,β₂ andβ₃. The wound healing rate waspositively correlated to TGF-β₃, but it was negatively correlated to TGF-β₁ andβ₂.â‘¢TGF-β₁,β₂ andβ₃ mainly expressed in the cytoplasm and the cell membrane of fibroblasts.MOD value was used and it accurately reflected the expression of the three indexes mentionedabove.â‘£The expression of TGF-β₁ andβ₂ in TGF-β₃ group was dramatically lower thanthose in the SD-Ag group and EGF group. And the wound healing rate of TGF-β₃ group wasmuch higher than SD-Ag group and EGF group. All those data suggested that transgenic TGF-β ₃ fibroblasts could not only effectively facilitate would healing but also reduce the excessivetissue fibrous degeneration by down-regulating the level of TGF-β₁ andβ₂ by means ofpersistently secreting TGF-β₃ protein in the wotmd surface.