| Human umbilical cord-mesenchymal stem cells(h UC-MSCs)have broad application prospects in the field of tissue regeneration and transplantation.As an important seed cell in regenerative medicine,it’s important to study the influence of exogenous substances on its proliferation activity and osteogenic differentiation ability.At the present time,researches have shown that some natural compounds can affect stem cells activity and differentiation.The main purpose of this paper is to study the effects of flavonoids and Monascus fermented crude substance on the proliferation activity and osteogenic differentiation of h UC-MSCs.First,we studied the effects of four flavonoids with similar structures: genistein,daidzein,biochanin and genistin on the proliferation activity of h UC-MSCs;Then,the genistein with the best proliferation effect was selected by CCK-8 test results to study its effect on the osteogenic differentiation of h UC-MSCs.Meanwhile,the effect of Monascus fermentation metabolites on the proliferation and osteogenic differentiation of h UC-MSCs was studied.Finally,the crude extract of the fermentation product of Monascus was added to the h UC-MSCs medium,and the process flow of the freeze-dried powder preparation of stem cells and the construction of the factory were designed.The main findings are as follows:1.Detecting the effects of genistein,daidzein,biochanin and genistin on the cell viability of h UC-MSCs with CCK-8 method.The results showed that genistein(0.7μM-14 μM)and daidzein(0.8 μM-16 μM)can significantly promote the proliferation of h UC-MSCs,and the optimal effect can be achieved by treating at the concentration of 0.7 μM and 16 μM for 36 hours,cell proliferation rate are 426% and 330%,respectively,which are extremely significant differences compared with the control group(P<0.01).Biochanin(0.028 μM-3.5 μM)and genistin(0.09 μM-4.6 μM)can also significantly promote the proliferation of h UC-MSCs(P<0.01),but the effect is weaker than that of genistein,so the genistein concentration was chosen to be 0.7 μM and treated for 36 h as the optimum condition,QPCR was used to detect the effect of genistein on the related genes in the proliferation pathway PI3K/Akt.The results showed that genistein can significantly up-regulate the m RNA expression levels of related genes PI3 K and Akt in the proliferation pathway PI3K/Akt pathway(P<0.01),compared with the control group.Meanwhile,the WB method was used to detect the expression of related proteins of Akt and P-Akt in the pathway,and the results showed that genistein can up-regulate the expression of P-Akt protein.2.To explore the effect of genistein(0.07 μM-7 μM)on the osteogenic differentiation of h UC-MSCs.The results of ALP activity in the early stage of osteogenic differentiation showed that: after induction for 14 days,each concentration of genistein could significantly enhance ALP activity compared with control group(P<0.01),which was concentration-dependent.After induction for 28 days,the calcium content in the late stage of osteogenic differentiation showed that the calcium content increased with the increase concentration of genistein.The 0.7 μM and 7 μM of genistein had extremely significant differences compared with control group(P<0.01).The staining results of Alizarin Red S stain showed that staining area increased and the degree of staining became deeper with the increase of genistein concentration.Quantitative analysis of the number of mineralized nodules observed in a randomly selected field of view under an inverted microscope(×100)found that the number of mineralized nodules increased with the increase of genistein concentration.The optimal concentration of 7 μM was selected according to the basic osteogenic indicators,and its effect on osteogenic differentiation-related genes was studied by QPCR.The results showed that the osteogenic-related genes Runx2,OCN,OPN,ALP,OSX m RNA were significantly up-regulated(P<0.01),after induced for 14 days.It is speculated that genistein may promote the osteogenic differentiation of h UC-MSCs by up-regulating the expression of osteogenesis-related genes.3.DMSO and ethanol were used as extraction solvents to obtain crude Monascus fermentation metabolites.The proliferation activity of h UC-MSCs was tested with CCK-8 method.The results showed that the drug group obtained by using DMSO as a solvent had better effects than ethanol.Therefore,DMSO was chosen as the solvent.After treated for 36 hours,the optimal concentration of co-fermentation group and blank fermentation group were 220+ μg/m L and 3.5 μg/m L,respectively.Meanwhile,the proliferation activities of h UC-MSCs were 230% and 314%,respectively.Compared with the control group,there was significant difference(P<0.01).The ALP activity test results showed that the fermentation metabolites of Monascus can enhance the ALP activity during the osteogenic differentiation of h UC-MSCs,and it is dosedependant.After treated with the concentration of 220+ μg/m L,220 μg/m L,the ALP activity were 21.19 Kings unit/gprot and 19.97 Kings unit/gprot,respectively,Which were significant different from the control group(P<0.05).The ALP activity of each concentration group of co-fermentation metabolites were higher than blank fermentation group.4.The plant for biological preparations with stem cell freeze-dried powder as the main ingredient was designed,and the equipment selection,process flow and plant construction for the annual production of 20,000 boxes of stem cell freeze-dried powder preparations were determined.It is estimated that the annual profit can reach 44.6million yuan,and the cost can be recovered after 1.1 years. |
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