【作者】 吕文天；

【导师】 龚守良； 李校堃；

【作者基本信息】 吉林大学 ， 放射医学， 2005， 博士

【摘要】 酸性成纤维细胞生长因子(aFGF)在机体生长发育中发挥重要的有丝分裂作用。aFGF还具有非促分裂作用,如神经保护和心肌缺血保护等。aFGF在体外发挥非促分裂作用时,可能会产生由促分裂活性诱导的细胞转化,从而有引起细胞非正常增殖而可能产生的潜在危险。 为了研究降低aFGF促分裂活性方法,从研究aFGF多肽链上的核转位序列与aFGF的信号途径的关联入手。通过人工敲除aFGF的该序列,制备了重组aFGF蛋白冻干粉(raFGF)。检测了两种蛋白的有丝分裂活性和受体酪氨酸激酶的信号途径。经比较分析认为,rFGF的促分裂活性明显低于waFGF。分析表明,raFGF的促分裂活性只与MAPK的级联有关,而不能够通过核转位途径发挥作用,而核转位序列在核转位途径中发挥重要作用。waFGF则可通过两种途径的协同作用发挥促分裂活性。对raFGF的细胞效应检测表明,raFGF具有维持细胞正常生存、避免发生细胞凋亡的作用。该研究结果在我们所做的动物实验中进一步得到的补充,即raFGF不但能够延长aFGF缺乏的新生大鼠的生存时间,而且还能够在一定程度上具有维持幼鼠正常神经系统功能的作用。而重要信号途径的丧失则有可能使raFGF的诱发细胞转化的可能性极小。更多还原

【Abstract】 Human acid fibroblast growth factor (aFGF) is the first member to be described of 23 closely related proteins in FGF family. aFGF is a very powerful and broad-spectrum mitogen. Actually, all the cells of mesodermal and neuroectodermal origin are differentiated by the stimulation of either aFGF or bFGF. The mitogenic activity induced by aFGF involves two signal pathways, one is Ras/MAPK cascade known as a classical signal pathway, the other is nuclear translocation process by which exogenous aFGF transports from the extracellular to the cytosol with the presence of functional residues like nuclear localization sequence (NLS) of aFGF polypeptide. Nuclear translocation process has been considered as an indispensable process in all cases for aFGF mitogenic activity.Besides the mitogenic activity, aFGF also has the nonmitogenicactivities in neuroprotection, vasodilatation and cardio-ischemia protection. When aFGF excerts its nonmitogenic activity, it will cause the possibly secondary effect derived from its mitogenic activity, such as cell transformation, carcinogenesis, etc. Aiming at making sure how to make aFGF lose or reduce its mitogenic activity and excert its nonmitogenic activity specially, the NLS of aFGF polypeptide was knocked out and prepared the recombinant aFGF (raFGF)o Through the comparation of wild-type aFGF (waFGF) and raFGF either in biological effects or in signal pathways, the mitogenic activity of w(r)aFGF were evaluated.1 Heating and frost thawing stabilities and sensitivity to tripsinTo assay the differences of biochemical character between raFGF and waFGF, we firstly compared their structural stability in conditions of heating, frost thawing and trypsin digestion by means of Western blot. The normal structure of aFGF was expressed with the target antigen structure which near the C-terminal of aFGF. For the heating stability test, the results showed no significant difference between raFGF and waFGF with heating for different time under 90°C. For the frost thawing stability, the results showed no significant difference between raFGF and waFGF with frost thawing for different time under -20°C. For the sensitivity to trypsin, the results showed that the band for raFGF digested with trypsin (0.025 g/ml) at 37℃ for 10 min was absent, but present in waFGF group. The results suggest that there is the significant difference between raFGF and waFGF under some special conditions.2 Cell proliferation assayTo test the mitogenic activity of both raFGF and waFGF as well asthe role of heparin in their activities, the cell proliferation assay was performed with MTT. This test could also determine the applied concentrations of raFGF, waFGF and heparin.2.1 Dose-effect relationship of heparinThe NIH3T3 cells were serum-starvation for 48 h in the presence of insulin and transferrin firstly, and then treated with raFGF and waFGF for next 48 h, the cell proliferation assay was performed. For the heparin, the dose-effect relationship was examined in the presence of various concentrations of wFGF. The results showed that the highest mitogenic activity occurred in waFGF groups when the concentrations of heparin were among 10-40 ng/ml. The results suggest that the heparin has the possibility to hence the activity of aFGF, and its applied concentration is 10 U/ml.2.2 Dose-effect relationship of raFGF and waFGFFor the cell proliferation assay for raFGF and waFGF, there were significantly different mitogenic activities between raFGF and waFGF when the concentrations were over 1 ng/ml. And waFGF excerted its activity with biggest degree when the concentrations were around 20 ng/ml. The results suggest that raFGF also has the mitogenic activity, but is significantly smaller than that of waFGF. Heparin can enhance the aFGF activity.3 aFGF-receptor tyrosine kinase phosphorylation and c-fos gene expressionThe signal pathways of both MAPK cascade and nuclear translocation are all involved in the activity of aFGF. With cellproliferation assay, the FGFR phophorylation and the c-fos gene response to stimuli are the important indexes related to the both pathways.3.1 aFGF-receptor tyrosine kinase phosphorylationFor the reason that NIH3T3 cells have FGFR1, we examined the FGR1 phosphorylation. NIH3T3 cells were stimulated for 8 min by either raFGF or waFGF, following serum-starvation for 48 h in the presence of insulin and transferrin. The results showed that there were clear binds for raFGF and waFGF, but no binds for without raFGF and waFGF. The results suggest that either raFGF or waFGF can induce the phosphorylation of FGFR1 tyrosine kinase.3.2 c-fos mRNA expressionOn the basis of phosphorylation and cell proliferation, to test whether there are difference in stimulating c-fos gene expression in order to judge the difference of signal pathways of raFGF and waFGF. The c-fos mRNA was expressed by mean of RT-PCR. The NIH3T3 cells were serum-starvation for 48 h in the presence of insulin and transferrin firstly, and then treated with raFGF and waFGF respectively, either in the presence or in the absence of heparin (10 U/ml). The electrophorotype showed that both raFGF and waFGF with and without heparin stimulated the c-fos gene expressions.As the miogenic activity of raFGF is expressed with the reference of cell proliferation, it was smaller than that of waFGF. We reasoned that waFGF excerts its function through both MAPK cascade and the nuclear translocation. Whereas the signal pathway for raFGF whichhas no NLS in its polypeptide is related to the MAPK cascade, but not to the nuclear translocation. 4 Cell cycle analysisTo test how raFGF and waFGF influence the cell cycle through examining both Giand G2 phase, now that both of them can affect cell proliferation. The analysis of 24 h-time-course was performed.4.1 Gi phase analysisNIH3T3 cells were incubated in 10% FCS for 24 h, and then serum-starved for next 48 h, and then treated with raFGF and waFGF with and without the presence of heparin for 24 h, respectively.The results showed that Gi phase arrest in almost all the cells occurred after 48 serum-starvation for 48 h. For the dose-effect relationship, the CP in raFGF + heparin, waFGF, and waFGF + heparin groups decreased firstly 9 h after serum-starvation, and then increased. On the contrary, the CP in raFGF group increased apparently within the first 9 hours, and then decreased. At 9 h, there was significant difference between the CP of raFGF and waFGF.The results suggest that the activity of raFGF is lower than that of waFGF, such can not reverse the Gi phase arrest immediately like what the waFGF do, and that heparin can enhance the raFGF activity.4.2 G2 phase analysisAlmost all the changes of CP in G2 phase for each test within 24 h treatment could not show. The results suggest that there is no significant G2 phase arrest happened in the effect of raFGF and waFGF on cell cycle do not affect the normal cells.Taken together, the cell cycle analysis suggests that waFGF could act as the positive role in sustaining the cell cycle progression through relieve Gi arrest; the activation of raFGF, which is less than that of waFGF, can play significantly the positive role only in the presence of heparin. With the reference of above-mentioned experimental results, we reasoned that the heparin is possibly the useful assistant factor when waFGF exerts its biological function in vivo.5 Apoptosis analysisThe effect of raFGF and waFGF on the apoptosis was tested in order to explain the biological activities of them.Upon the dose-effect relationship, in general, the CP in all the groups decreased at the first 12 h. And then, the CP in both r and rh groups almost went flat until 18 h at which the CP increased. The CP in w and wh groups decreased from 12 h later. The most significant changes of the CP in rh and wh groups in 24 h-time-course appeared from 12 to 18 h to show an opposed tendencies.The apoptosis test suggests that both waFGF and raFGF cause the decreased apoptotic CP, but the latter only act for very short time. We reason that the decreased CP might be due to the inhibition of waFGF or raFGF on apoptosis through the mitogenic activity. With the participant of heparin, either waFGF or raFGF show much more inhibition activity.6 Assays for p53 mRNA and Bcl-2 mRNA expressionsIn the assay for apoptosis, the raFGF could cause the apoptosis in the latter stage of 24 h -time-course, whereas raFGF showed the weakmitogenic activity in other tests. Thus, it was necessary to test the relevant gene expression to analysize the paradox results. p53 gene and Bcl-2 gene are the closely related to the outset of apoptosis. NIH3T3 cells were incubated in 10% FCS for 24 h, and then serum-starvated for next 48 h, and then treated with test compounds for 9 h. With the reference of gene-GAPDH gene expression, the comparison of various mRNA levels was performed.The p53 mRNA in r group was significantly higher than that in w group, either in the presence or in the absence of heparin. On the contrary, Bcl-2 mRNA in r group was significantly lower than that in w and wh groups, either in the presence or in the absence of heparin. We reason that the positive p53 gene expression in w and wh groups should be due to the normal apoptosis with no concern of waFGF. It was normal condition for the weak expression of Bcl-2 gene in r and rh groups. 7 Cell membrane permeability analysisTo test the relationship of aFGF and apoptosis, it is essential to study the effect of aFGF on apoptosis-related membrane permeability. The part procedure of cell membrane permeability analysis was the same as the assay for cell cycle, and was expressed with the percentage of cells dyed by PI.The results showed that almost all the CP in various groups decreased within 18 h. And then went stable for waFGF and waFGF + heparin groups, and increased to some extend for raFGF and raFGF + heparin groups. The CPs In the raFGF group with and without thepresence of heparin, there were none of significant difference. There were the significant differences for the comparisons of CPs in waFGF and raFGF groups as well as waFGF and waFGF + heparin groups. The results suggest that the capability of cell membrane permeability sustained by raFGF is positive. 8 Mitochondria membrane potential testThe mitochondria membrane potential was another important reference to reflect the state of cell survival. The part procedure of mitochondria membrane potential test was the same as the assay for cell cycle. At the end of treatment, cells were dyed with Rhl23 at 37"C for 30 min. Mitochondria membrane potential was analysized with FCM and expressed as the percentages of cells dyed by Rhl23. 8.1 Mitochondria membrane potential testFor the 24 h-time-course, almost all the mitochondria membrane potentials in all groups tend to go down at the first 9 h in the experiment with different cell percentages, and then increased continually. As the comparison for each two groups, there were the significant differences of CP in paired r-wh and rh-wh groups at 6 h. And the continued significant differences of CP in this two paired groups almost showed in the following time courses except at 9 h for paired rh-wh group. From 9 h later, the CP in all the groups went upward. There was the significant difference of CP in the paired r-w group at 12 h, at 15 h for w-wh and w-rh groups, and at 21 h for r-rh group.The results suggest that raFGF can keep the mitochodriamembrane potential to some extend. With the references for cell membrane permeability assay, p53 and Bcl-2 gene expressions and apoptosis analysis, the results suggest that the mitogenic activity of raFGF, although lower than that of waFGF, can sustain the normal cell survival, such as normal cell membrane permeability and mitochodria membrane potential. This activity can be enhanced by heparin. If this activity is connected with the results of apoptosis analysis, we can reason that the time for raFGF-induced cellular protective activity is around 12-15 h, whereas the time for waFGF is much more 24 h. And this may be due to the different structural stability. 8.2 Cell observation under fluorescence microscope.We could observe the NIH3T3 cells dyed with either Rhl23 or PI or both of them under the fluorescent microscope aiming at abserve the state of cell survival. The red cells wholly belonged to the state of necrosis or certain state of cells in which the mitochondria do not work anymore. The green cells wholly belonged to normal cells. The cells with both red and green colours belonged to the state of apoptosis. 9 Test in vivoSuramin is the antagonist of aFGF. The animol model of the new-born rats of aFGF-lacked was made by means of suramin (10 mg/kg) injection, and at the same time, the rats were compensated with exogenous either raFGF or waFGF (100 ug/kg), respectively. Then the effects of either raFGF or waFGF on rats were examined through the observations of body weight, external appearance, time for opening eyes and survival rate.Five treatment groups: suramin, suramin + waFGF, suramin + raFGF, normal, blank (physiologic saline).9.1 Time-course test of body weightThe arrange of various groups in the speed of body weight increasing in from high to low was normal, blank, suramin + waFGF, suramin + raFGF and suramin groups, respectively. All the rats in s group died within 16 d after birth. The comparisons of body weight in various groups at 8, 12 and 16 d after birth showed that there were significant differences among these groups except that in suramin and suramin + raFGF groups at 8 d after birth.9.2 External appearanceFor suramin group, the desquamate on the backs in 15 d old rats appeared. CNS disfunction symptom — opisthotonos with frequemcy of (1 ~ 2 times/h) in 13 d old rats in s group appeared.9.3 Survival rateThe first case of death appeared at 12 d old rats in suramin group in which all rats died within 16 d. The survival rate in suramin + raFGF group was significantly lower than that for suramin + waFGF group. Survival rates in both suramin + raFGF and suramin + waFGF groups were lower than those in normal and blank groups.9.4 Time for opening eyesThe time for opening eyes was at 14 d in normal group, at 19 d in suramin + waFGF group and at 22 d in suramin + raFGF group, respectively. All the rats in s group died before opening eyes.The results suggest that the ability of raFGF on inhibiting suramin更多还原