［摘要］目的：重组碱性成纤维细胞生长因子（rhbFGF）对培养条件下3T3成纤维细胞在血纤维蛋白凝块上生长的可能性研究。方法：采用Giemsa染色和MTT检测法，经过电镜扫描，分段对比观察，研究3T3细胞在血纤维蛋白凝块上的生长情况。 结果：rhbFGF促进细胞生长并维持细胞存活的最佳浓度是100 ng/mL，在含有100 ng/mL的低血清培养基中细胞可在血纤维蛋白凝块上生长。经48h培养以后仍有大量的细胞存活。 结论：3T3成纤维细胞可在含有rhbFGF的低血清培养基上生长并存活，rhbFGF与血纤维蛋白凝块共存可以促进创伤愈合。

［中图分类号］Q 813［文献标识码］ A

［Article ID］1000-4718(2000)02-0097-05

Possibility of 3T3 fibroblast growth on blood fibrin clot in culture medium with recombinant human basic fibroblast growth factor

WANG Yi-fei, DAI Yun, LIU Jie-shen, LIN Jian

(Bioengineering Institute of Jinan University, Guangzhou 510632, China)

CUI Yun-xia

(The Research Center of Molecular Medicine, Sun Yet-Sen University of Medical Sciences,Guangzhou 510089, China)

［Abstract］ AIM: To investigate the possibility of 3T3 fibroblast growth on blood fibrin clot in culture medium with recombinant human basic fibroblast growth factor (rhbFGF).METHOD: Growth of the cells on blood fibrin clot was studied by phase-contrast, scanning and transmission electron microscopy and by Giemsa stain and MTT assay.RESULTS: The optimal concentration of rhbFGF for proliferation and survival of the cells was 100 ng/mL. The cells also grew on blood fibrin clot scaffold in the low-serum medium containing 100 ng/mL rhbFGF, and a greater number of the cells survived after 48 hours incubation compared to that after 24 hours. The elongated filopodia appeared to bridge the gaps among the fibroblasts after 24 hours incubation. Further incubation to 72 hours, a greater number of platycytes were found to be joined together by lamellopodia.CONCLUSION: 3T3 fibroblasts could grow and survive on blood fibrin clot in the low-serum medium containing rhbFGF, and a combination of blood fibrin clot and rhbFGF may have over proportional effects on wound healing.

［MeSH］Fibroblasts;Agglutination;Fibroblast growth factor, basic

［CLC number］Q813［Document code］A

INTRODUCTION

Fibrin clot obtained from fresh blood is a kind of biomaterial with low immunogenicity or no antigenicity, usually acting as a physiological scaffold in wound healing^［1～4］. rhbFGF is an active peptide growth factor capable of facilitating the proliferation and differentiation of the majority of cells derived from the mesoderm and neuroectoderm as well as promting processes in wound healing^［5～8］. A series of investigation concerning the effect of rhbFGF on the formation of three-dimensional tissue model has been made and we here report the possibility of 3T3 fibroblasts on blood fibrin clot immersed in a low-serum medium supplemented with rhbFGF.

MATERIALS AND METHODS

Experimental animals and reagentsBALB/C mice aged 8～12 weeks were used. Trypsin, penicillin, streptomycin, fetal bovine serum and Dulbecco's Modified Eagle Medium (DMEM) were purchased from Giebco Ltd., Paisley, UK; MTT ［3-(4, 5-dimethylthiazal-2yl)2,5-diphenyltetrazolium bromide］ was a product of Sigma Chemical Co., St. Louis, MO; rhbFGF (No. 199806) was a purified gene recombinant product of Bioengineering Institute of Jinan University.

Preparation of blood fibrin clot The eyeballs of six BALB/C mice were ablated with an eye-tweezers under a relatively aseptic condition, and the blood collected was let to stand for 30 minutes in a centrifuge tube. The serum was then removed after centrifugation by 1 500 r/min for 10 minutes. The blood fibrin clot thus obtained was washed repeatedly with double-distilled water until it became free of red color, and then twice with Hanks solution containing penicillin (50 U/mL) and streptomycin(50 μL/mL). The blood fibrin clot was subsequently cut into a number of pieces which were put separately into the centre of a series of culture flasks to be lyophilized in a cryodesiccator (HETOTR^ CT-60-e) for 2 hours.

Cell culture3T3 fibroblasts were cultured in DMEM containing 10% (vol/vol) fetal bovine serum and penicillin (50 U/mL)/streptomycin ( 50 μL/mL). When the bottom of the flasks was nearly packed with confluent cells, 0.1% (wt/vol) trypsin was used to detached the fibroblasts at 37 ℃ for 3 minutes. The dissociated cells were then washed and resuspended in DMEM with 10% (vol/vol) fetal bovine serum. Concentration of the cell suspension was calculated and adjusted to 5×10⁵/mL.

MTT assay^［9,10］ Cell suspensions (5×10³/mL) containing 10%(vol/vol) fetal bovine serum in DMEM were seed onto 96-well flat-bottomed plates (100 μL/well) and incubated 24 hours ,the culture medium was removed , the cells were then washed with phosphate buffered saline three times and re-fed with 100 μL/well of 0.4% (vol/vol) fetal bovine serum in DMEM containing a series of different dilutions of rhbFGF for further incubation. Baseline control (100 μL/well fetal bovine serum in DMEM) parallel to the sample for each plate. 48 hours after the further incubation , 10 μL MTT solution (5 mg/mL) was added immediately to each well of the assay and the plates were incubated at 37 ℃ for another 4 hours. Acid-isopropanol (100 μL of 0.04 mol/L in isopropanol) was added to each well and the contents of which were mixed thoroughly to dissolve the dark blue crystals. The plates were let to stand for a few minutes at room temperature to ensure complete dissolution of the crystals . The OD vaule of each well of the 3T3 fibroblast suspensions incubated with different concentrations of rhbFGF was read out on an EL311 autoplate reader (BIO-TEK Instruments) with a test wavelength of 570nm and a reference wavelength of 630nm, so that the optimal concentration of rhbFGF for stimulation of the proliferation of 3T3 fibroblasts could be determined.

Phase contrast microscopic observation 5 mL of cell suspension (1.5×10⁵/mL) in DMEM with 10%(vol/vol) fetal bovine serum were added separately to each of the 25 cm×25 cmculture flasks with and without blood fibrin clot specimen. Incubated 24 hours, the medium containing 10%(vol/vol) fetal bovine serum was removed out from the culture flasks and the behavior of the cells plated on the blood fibrin clots or glasses was monitored and photographed under a phase contrast microscope. Subsequently, 5 mL each of fresh DMEM containing 10% (vol/vol) fetal bovine serum were added separately into half of the flasks while another half of the flasks were added 5 mL each of fresh DMEM containing 0.4%(vol/vol) fetal bovine serum supplemented with rhbFGF in an optimal concentration as shown in the MTT assay. The growth behaviour of 3T3 fibroblasts plated on blood fibrin clot in each of the flasks was monitored and photographed under the phase contrast microscope after 24 hours as well as 48 hours of incubation, respectively, so as to compare the behaviour of the cells grown in the two different culture media.

Giemsa stainAfter 72 hours of incubation, the culture medium in each of the flasks was quikly removed out and the cells were washed three times with 0.1 mol/L PBS, fixed in cold methanol:glacial acetic acid (3∶1) for 30 minutes, washed twice in 0.1 mol