含重組堿性成纖維細胞生長因子培養(yǎng)基中3T3成纖維細胞在血纖維蛋白凝塊上生長的可能性

1、    含重組堿性成纖維細胞生長因子培養(yǎng)基中3T3成 纖維細胞在血纖維蛋白凝塊上生長的可能性        摘要目的：重組堿性成纖維細胞生長因子（rhbFGF）對培養(yǎng)條件下3T3成纖維細胞在血纖維蛋白凝塊上生長的可能性研究。方法：采用Giemsa染色和MTT檢測法，經(jīng)過電鏡掃描，分段對比觀察，研究3T3細胞在血纖維蛋白凝塊上的生長情況。 結(jié)果：rhbFGF促進細胞生長并維持細胞存活的最佳濃度是100 ng/mL，在含有100 ng/mL的低血清培養(yǎng)基中細胞可在血纖維

2、蛋白凝塊上生長。經(jīng)48h培養(yǎng)以后仍有大量的細胞存活。 結(jié)論：3T3成纖維細胞可在含有rhbFGF的低血清培養(yǎng)基上生長并存活，rhbFGF與血纖維蛋白凝塊共存可以促進創(chuàng)傷愈合。主題詞成纖維細胞；凝集；成纖維細胞生長因子，堿性中分類號Q 813文獻標識碼 AArticle ID1000-4718(2000)02-0097-05 Possibility of 3T3 fibroblast growth on blood fibrin clot in culture medium with recombinant human basic fibroblast growth factorWANG Yi-

3、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 o

4、n 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

5、 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 appe

6、ared 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,

7、 and a combination of blood fibrin clot and rhbFGF may have over proportional effects on wound healing.MeSHFibroblasts;Agglutination;Fibroblast growth factor, basic CLC numberQ813Document codeAINTRODUCTIONFibrin clot obtained from fresh blood is a kind of biomaterial with low immunogenicity or no an

8、tigenicity, usually acting as a physiological scaffold in wound healing14. 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 healing58. A

9、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 METHODSExperimental animals and re

10、agentsBALB/C mice aged 812 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. Lou

11、is, 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

12、 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) an

13、d streptomycin(50 L/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% (vo

14、l/vol) fetal bovine serum and penicillin (50 U/mL)/streptomycin ( 50 L/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% (v

15、ol/vol) fetal bovine serum. Concentration of the cell suspension was calculated and adjusted to 5×105/mL.MTT assay9,10 Cell suspensions (5×103/mL) containing 10%(vol/vol) fetal bovine serum in DMEM were seed onto 96-well flat-bottomed plates (100 L/well) and incubated 24 hours ,the culture

16、 medium was removed , the cells were then washed with phosphate buffered saline three times and re-fed with 100 L/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 L/well fetal bovine serum in DMEM) pa

17、rallel to the sample for each plate. 48 hours after the further incubation , 10 L 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 L of 0.04 mol/L in isopropanol) was added to each well and the conte

18、nts 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

19、 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 cel

20、l 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 flas

21、ks 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 fla

22、sks 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

23、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

24、 three times with 0.1 mol/L PBS, fixed in cold methanol:glacial acetic acid (31) for 30 minutes, washed twice in 0.1 mol/L PBS again and then observed under the phase contrast microscope after Giemsa staining. 3T3 fibroblasts were stained blue in Giemsa staining, whereas blood fibrin clots, light re

25、d in color, thus rendering the latter to be readily distinguished from the former . With the increase in the number of the fibroblasts, the colour of the cells turned from light to deep blue.Scanning electron microscopic observation 3T3 fibroblasts with both blood fibrin clot and glass control as su

26、bstratum were fixed successively in 2% glutaraldehyde in 0.1 mol/L PBS and osmium cacodylate in 0.1 mol/L PBS. The specimens were then dehydrated through a grade series of acetone and water, and dried with CO2 and gold sputter-coated for examination under a scanning electron microscope (JEOL JSM-730

27、0).Transmission electron microscopic observation 3T3 cell specimens with blood fibrin clot as substratum were fixed successively with 2% glutaraldehyde in 0.1 mol/L PBS at 4 and 1% osmium tetraoxide for 2 hours. Ultrathin sections were stained with 2% uranyl acetate and lead citrate and were inspect

28、ed under a transmission electron microscope (JEOL 100×II/T).RESULTS AND DISCUSSIONEffect of rhbFGF on 3T3 fibroblast proliferation and survival To evaluate the effect of rhbFGF on 3T3 fibroblast proliferation and survival, it is necessary to observe the impact of rhbFGF in different concentrati

29、ons on the cells.It was shown that rhbFGF in concentrations higher than 25 ng/mL displayed a promotive effect on the proliferation of the cells incubated in DMEM with 0.4% (vol/vol) fetal bovine serum, and this effect was gradually augmented with increasing concentrations of rhbFGF. rhbFGF in still

30、higher concentration, ie 800 ng/mL, however, depressed the proliferation of the cells. The optimal concentration of rhbFGF for the promotion of cell proliferation was found to be 100 ng/mL (P0.01). The results also showed that 3T3 fibroblast adhered and grew well on blood fibrin clot bathed in DMEM

31、containing 0.4% (vol/vol) fetal bovine serum supplemented with 100 ng/mL rhbFGF, indicating that rhbFGF can be used as an important component of a novel low-serum or serum-free medium for the growth of fibroblasts (Tab 1).Tab 1OD of 3T3 fibroblasts in culture media with different concentrations of r

32、hbFGF in MTT assay(±s, n=12)rhbFGF concentration (ng/mL)Control800400100256.253.1251.5620.103±0.002*0.138±0.0050.152±0.0050.140±0.0080.130±0.008*0.129±0.007*0.130±0.008*0.115±0.011*P0.05, P0.05, P0.01, vs control 3T3 fibroblast growth on blood fibrin clot

33、 3T3 fibroblasts grew well on blood fibrin clot in DMEM supplemented 10% (vol/vol) fetal bovine serum, as shown under the phase-contrast microscope (Fig 1). The cells also can grew on blood fibrin clot scaffold in the low-serum containing 100 ng/mL of rhbFGF (Fig 2), and a significantly greater numb

34、er of 3T3 fibroblasts were seen to grow on blood fibrin clot after 48 hours of incubation as compared with these after 24 hours of culture in the low-serum DMEM(Fig 3). These results suggest that the blood fibrin clot may serve not only as a support system, but also a carrier of rhbFGF in wound heal

35、ing.3T3 fibroblast phenotypes on blood fibrin clot A number of high resolution images of the key behavioural phenotypes of 3T3 fibroblasts grown on blood fibrin clot or glass at different intervals of incubation were shown by scanning electron microscopy. Elongated filopodia were seen to bridge the

36、gaps among 3T3 fibroblasts that had been incubated for 24 hours on blood fibrin clots bathed in DMEM containing 0.4% (vol/vol) fetal bovine serum supplemented with 100 ng/mL of rhbFGF (Fig 4). After 72 hours of incubation, a great number of platycytes were found to be joined together by lamellopodia

37、 (Fig 5). Besides, 3T3 fibroblassts could grow and spead well on both blood fibrin clot and glass (Fig 4, Fig 6).Fig 13T3 fibroblast culture on blood fibrin clot bathed in 10% fetal bovine serum for 24 hours×150Fig 2 3T3 fibroblast culture on blood fibrin clot bathed in 0.4% fetal bovine serum

38、DMEM supplemented 100 ng/mL rhbFGF for 24 hours×150Fig 3 3T3 fibroblast culture on blood fibrin clot in 0.4% fetal bovine serum DMEM supplemented 100 ng/mL rhbFGF for 48 hours×150Fig 4 3T3 fibroblasts growth on blood fibrin clot bathed in DMEM containing 0.4% fetal bovine serum supplemente

39、d with 100 ng/mL of rhbFGF for 24 hoursThe structure of 3T3 fibroblasts and blood fibrin clotTransmission electron microscopic examination revealed that 3T3 fibroblasts could well adhere to and on the surface of blood fibrin clot. Besides, blood fibrin clot was found to possess a large number of min

40、ute pores through which adequate nutrients can be readily supplied to the cells (Fig 7).In conclusion, as a non-antigenic derived from fresh blood, fibrin clot may serve as a physiological support system and carrier of cellular growth factor rhbFGF to promote wound healing. We envisage that combined

41、 application of blood fibrin clot and rhbFGF should have a prospect in clinical medicine, notably in the treatment of traumas and wounds. The combination of blood fibrin and rhbFGF may also serve as a useful tool in the study of three-dimensional tissue culture, and the biological activity of rhbFGF

42、 should also be utilized to develop an economical and feasible low-serum or free-serum three dimentional tissue culture medium.Fig 5 3T3 fibroblasts growth on blood fibrin clot bathed in DMEM containing 0.4% fetal bovine serum supplemented with 100 ng/mL of rhbFGF for 72 hoursFig 6 3T3 fibroblasts g

43、rowth on glass bathed in DMEM containing 0.4% fetal bovine serum supplemented with 100 ng/mL of rhbFGF for 24 hoursFig 7 The structure of 3T3 fibroblast growth on blood fibrin clot for 24 hours×4031Foundation itemSupported by Grant (No. 96-C02-01-03) from the Key Scientific Item in National “9.5” of China王一飛（暨南大學(xué)生物工程研究所，廣東 廣州 510632）戴云（暨南大學(xué)生物工程研究所，廣東 廣州 510632）劉杰生（暨南大學(xué)生物工程研究所，廣東 廣州 510632）林劍（暨南大學(xué)生物工程研究所，廣東 廣州 510632）崔蘊霞（中山醫(yī)科大學(xué)分子醫(yī)學(xué)研究