Effect Of Diabetic State On The Functional Activity Of Circulating Peripheral Blood Fibrocytes

Part Ⅰ Isolation, Cultivation and Identification of Circulating Fibrocytes of Diabetic PatientsObjective:To discuss the method to isolate and cultivate peripheral blood circulating fibrocytes (cFb) in diabetic patients and to explore the conditions to carry out subsequent researches on the biological activity of cFb in burn patients.Methods:Peripheralblood mononuclear cells in burn patients were isolated by density gradient centrifugation method and then inoculated into Dulbecco’s Modified Eagle Medium (DMEM) containing 20% of fetal calf serum (FBS) for 28 days of continuous cultivation. Except rising with poly butylenes succinate (PBS), no special treatment was given each time DMEM was changed.The cells isolated were observed under inverted fluorescent contrast phase microscope on the same day of isolation and inoculation, the 3th day, the 5th to 6th day, 7th to 8th day,14th day,21th day,24th day and 28th day in terms of cell proliferation and morphological change. Also, their pictures were taken by a digital camera.The abilities of the adherent cells isolated and cultivated for 14 and 28 days to express extracellular matrix proteinCol-Ia, a-SMAand growth factors TGF-β1 and bFGF were detected by real-time fluorescent quantitative PCR to carry out functional identification.Results:(1) Morphologically, PBMCs isolated from the peripheral blood of diabetic patients were spheroidal. After isolation and cultivation for two or three days, most of PBMCs were still sporadic, suspendedballs, but partial sporadic cell colony formation was noted, with a small amount of fusiform adherent cells in the margin. And the cell colony was much more obvious and the number of fusiform cells was increased by the 4th day. Later, the cell colony was reduced around the 5th to 7th day. On the 10th day, the cell colony totally disappeared and was substituted by the proliferation of fusiform cells which were regularly arrayed with the appearance of typical fusiform fibrocytes. On the 14th day after isolation, the density of these fusiform cells was increased and they were regularly arranged and displayed typical fusiform. On the 21th day after isolation, rapid proliferation could be detected, and these cells were regularly arrayed and displayed typical fusiform. On the 28th day after isolation, cell proliferation was observed and these cells were regularly arranged in typical fusiform shape.(2) cFb in diabetic patients cultivated in vitro for 28 days showed higher expressions of Col-la and a-SMAmRNA. And the expression level of Col-la mRNA was 72716.74 times of that on the 14th day (P<0.05), while the expression level of a-SMAmRNA was still increased to 2.38 times of that on the 14th day, but there was no statistically significantdifference (P>0.05).(3) cFb in diabetic patients cultivated for 28 days showed lower ability to express TGF-β1 mRNA compared with that cultivated in vitro for 14 days, but there was no statistically significantdifference (P>0.05); while the relative expression level of bFGF mRNA was obviously increased to 5.98 times of the former (P<0.05).Conclusion:With the application of density centrifugation-adherent culture method, it is possible to successfully isolate cFb with highly expressed mesenchymal cell makers Col-la and a-SMA mRNA in typical fusiform shape from the peripheral blood of diabetic patients.Part Ⅱ Effect of Diabetes Mellitus on the Activity of PBMCs in Expression CytokinesObjective:Through a comparative study on the circulating fibrocytes (cFb) in diabetic patients and healthy subjects, the objective of the present study is to observe the two group’s abilities to synthesize ECM Col-Iα, α-SMA, chemokineMIP-1α receptors CCR5 and CXCR4, inflammatory mediator TNF-α and IL-6, and growth factors TGF-β1 and bFGF mRNA, to understand the effect of diabetes mellitus on the biological activity of cFb represented by the ability to express cytokine and to provide bases for further clinical application studies.Methods:Peripheral blood mononuclear cells (PBMCs) in burn patients were isolated by density gradient centrifugation method and then inoculated into Dulbecco’s Modified Eagle Medium (DMEM) containing 20% of fetal calf serum (FBS) for 28 days of continuous cultivation. Except rising with poly butylenes succinate (PBS), no special treatment was given each time DMEM was changed. The success rate of isolation, cFb obtained rate and proliferation activity were observed on the 14th day and 28th day of isolation and cultivation.The abilities of cFbisolated and cultivated for 14 and 28 days to express ECM Col-Iα, α-SMA, chemokineMIP-1α receptors CCR5 and CXCR4, inflammatory mediator TNF-α and IL-6, and growth factors TGF-β1 and bFGF mRNA were detected by real-time quantitative PCR, and the abilities of diabetic patients and healthy subjects to express the mRNAs of the relevant factors at the above two time points were compared.Results:(1) In the process of isolated culture of cFb in peripheral blood,45 heparin anticoagulant specimens from healthy subjects and 47 anticoagulant heparin anticoagulant specimens from diabetic patients were enrolled into this study for isolation and cultivation in vitro. The results showed that 43 specimens of PBMCs (95%) were successfully isolated and 25 specimens of cFb (55%) were obtained in the normal group; while 43 specimens of PBMCs (91%) were successfully isolated and 25 specimens of cFb (32%) were obtained in the normal group. The samples of adherent fusiform cells obtained from isolation were observed for the proliferation activity in vitro. The result indicated that the cell fusion rate of cFb in healthy subjects cultivated for 5 to 7 days could reach about 50%, but the proliferation of cFb in diabetic patients was significantly delayed andrequired 10 to 14 days of cultivation in vitro to achieve a cell fusion rate of about 50%.(2) After cultivation in vitro for 14 days, the relative expression levels of ECM Col-la, chemokine MIP-1α, growth factor bFGF mRNAs in cFbof diabetic patients were higher than those of healthy subjects at the same time point, showing statistically significant differences (P<0.05); while the relative expression levels of ECM a-SMA, chemokine receptor CXCR4, inflammatory mediator IL-6, growth factor TGF-β1 mRNAs in cFb of diabetic patients were lower than those of healthy subjects at the same time point, but there were no statistically significant differences (P>0.05); the relative expression levels ofchemokine receptor CCR5, inflammatory mediatorTNF-a mRNAs in cFb of diabetic patients were similar with those ofhealthy subjects at the same time point, but there were no statistically significant differences (P>0.05).(3) After cultivation in vitro for 28 days, the relative expression levels of ECM Col-Iα and growth factor bFGF mRNAs in cFb of diabetic patients were higher than those of healthy subjects at the same time point, showing statistically significant differences (P<0.05); the relative expression levels of chemokine MIP-1α, chemokine receptor CXCR4, inflammatory mediator IL-6 mRNAs in cFb of diabetic patients were lower than those of healthy subjects at the same time point, showing statistically significant differences (P<0.05); the relative expression levels of ECM a-SMA, chemokine receptor CCR5, inflammatory mediator TNF-α and growth factor TGF-β1 mRNAs in cFb of diabetic patients were lower than those in healthy subjects at the same time point, but there were no statistically significant differences (P>0.05).Conclusions:(1) cFb from the peripheral blood of diabetic patients has lower success rate, obtained rate, proliferation activity and other indicators than the control group.(2) After cultivation in vitro for 14 days and 28 days, cFb in diabetic patients shows lower ability in expressing ECM α-SMA, inflammatory mediator and growth factor TGF-β1 mRNAs than cFb in healthy subjects, suggesting that diabetic state can inhibit the biological activity of cFb represented by the ability to express trauma related factors, thus affecting its clinical application value in diabetics and other chronic wound repair.(3) After cultivation in vitro for 14 days and 28 days, cFb in diabetic patients shows strongerability in expressing ECM Col-Iα and growth factor bFGFmRNAs than cFb in healthy subjects, suggesting thatcFbunder diabetic state improves its ECM synthesis and the expression level of neovascularization regulatory factor mRNA. This might promote the chronic wound repair of diabetic patients.