摘要
胸腺素是广泛存在于人体及动物体内的一组极性小分子多肽,可分成α及β型。β型胸腺素家族成员之间序列高度保守,分子量大多在5kDa左右。β胸腺素在细胞内的表达水平与细胞的分化程度密切相关。现在已知,β型胸腺素的主要功能是在细胞内与肌动蛋白单体以1:1的比例结合形成复合体,从而抑制肌动蛋白聚合的发生。家族成员中的胸腺素β4在组织中含量最多,分布最广,已经发现该蛋白具有多种生物学功能,如对金属蛋白酶活性、趋化作用、血管生成的诱导作用以及对炎性反应和骨髓干细胞增殖的抑制作用等等。血管的生成是伤口愈合的重要前提之一。由于Tβ_(16)的氨基酸序列与Tβ_4有着高度的同源性,所以我们通过人工合成获得Tβ_(16)小肽,设计促进血管增生实验、伤口愈合实验和划痕闭合实验等实验方法,以期得到该小肽在促进血管生成和创伤愈合方面的作用的结果。
Theβ-thymosins are a family of highly conserved polar 5 kDa peptides. They are present in almost every cell. Theβ-thymosins bind monomeric actin in a 1:1 complex and act as actin buffers, preventing polymerization into actin filaments but supplying a pool of actin monomers when the cell needs filaments. Changes in the expression ofβ-thymosins appear to be related to the differentiation of cells. Among the members of the family, thymosinβ4 is demonstrated to show many physiological effects, such as the induction of metallo-proteinases, chemotaxis, angiogenesis and inhibition of inflammation as well as the inhibition of bone marrow stem cell proliferation. Since thymosinβ16 is highly homologous with thymosinβ4, we concentrate our interest on the possible familiar effect of thymosinβ16 with thymosinβ4.
Purpose
To evaluate the effects of Tβ16 in promoting angiogenesis and wound healing.
Methods
The peptide thymosinβ16 was synthesized by Chinese Peptide Company.
1.“scratch”wound closure assay
Confluent HUVEC monolayers in 24-well plates were“scratch”wounded using the tip of a universal blue pipette tip and rinsed withD-Hank’s. Tβ_(16) (1μg/ml in DMEM with 10% fetal bovine serum) was add to six wells and the plates were incubated for 72 hours. And DMEM with 10% fetal bovine serum only and bFGF (1μg/ml in DMEM with 10% fetal bovine serum) were applied for control. The width of the wound was observed using a microscope with a 10X objective.
2. In vivo migration and angiogenesis assay
Fertile chicken eggs were obtained, and they were placed inside an incubator, at 373C.On the seventh day of incubation, a `window' (1.5×1.5 cm) was opened on the eggshell exposing the CAM. Tβ_(16) (50μg/ml in 50μl NS) were add to sterile plastic discs (of 5 mm diameter) and the discs were placed on the CAM area. NS only and aFGF (5μg/ml in 50μl NS) were applied for control. Three eggs were used for each group. The window was covered with sterile cellophane tape and the eggs were returned to the incubator where they were kept until day 9 for observation.
3. wound healing assay
One full thickness 3mm diameter punch biopsy wounds were made on the dorsal surface of each mouse. Tβ_(16) (0.5μg/μl in 50μl PBS) was applied topically on the second day, and again after 48 h to the wounds. At day 7, post-wounding, wound tissue was collected and fixed in formalin. The samples were sectioned, and sections from the middle of the wound were stained with H & E. PBS only and bFGF (0.5μg/μl in 50μl PBS) was applied for control. Histological sections were used to observed the re-epithelialization and the contraction of the wound using a microscope with a 4X objective.
4. wound healing following alkali injury assay
Six 18mm diameter alkali injury were made on the dorsal surface of each rabbit. Tβ_(16) (5,25,50 and 100μg/g in 0.5g hydrogel) was applied topically on the second day, and again after 48 h to the wounds. At day 15, post-wounding, wound tissue was collected and fixed in formalin. The samples were sectioned, and sections from the middle of the wound were stained with H & E. Hydrogel only and bFGF (4μg/g in 0.5g hydrogel) was applied for control. Histological sections were used to observed the re-epithelialization and the contraction of the wound using a microscope with a 4X objective.
Resullts
1. After 72 h the“scratch”wounds in four of the total six wells(Tβ_(16) 1μg/ml in DMEM with 10% fetal bovine serum) closed completely, none of the six wells(DMEM with 10% fetal bovine serum only) closed and three of the six wells(bFGF 1μg/ml in DMEM with 10% fetal bovine serum) closed completely.
2. The difference of the eggs treated with Tβ_(16) (50μg/ml in 50μl NS) and NS only was significant. The density of blood vessel on the CAM treated with Tβ_(16) were significantly higher than the three eggs treated with NS only.
3. Tβ_(16) (0.5μg/μl in 50μl PBS) promotes wound closure as observed by the increase in keratinocyte migration from the wound edges. The control mice showed little wound repair with poor formation of granulation tissue.
4. At day 15, post-wounding ,all the wounds treated with Tβ_(16) ( 20,30,50,100μg/g in 0.5g hydrogel) recovered , but the wounds treated with hydrogel only and bFGF ( 4μg/g in 0.5g hydrogel ) recovered incompletely.
Conclusion
1. Tβ_(16) promote HUVEC migration in vitro.
2. Tβ_(16) stimulate angiogenesis in the CAM model.
3. Tβ_(16) promote wound healing in C57 mice.
4. Tβ_(16) promote wound healing following alkali injury in rabbits.
In conclusion, it is well certified that the Tβ16 play an important role in promoting angiogenesis and wound healing through our works. Although it remains unknown that what mechanism Tβ16 follows, it seems obvious that Tβ16 may undergo such a change of functions from stimulating the migration of HUVECs to promoting the angiogenesis in wound healing. We make it sure that Tβ16 is valuable in clinic therapy in wound healing. And one of the major goals is to search for the molecular mechanisms mediating the effects attributed to extracellular Tβ16.
引文
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