| Objective: The heparin-hyaluronic acid-submandibular gland acellular matrix hydrogel was used for three-dimensional culture of submandibular gland cells in vitro,and their functional expression was preliminarily tested,laying the foundation for the construction of salivary gland organoids.Methods:(1)Enzymatic digestion method was used to obtain SD rat submandibular gland cells and perform immunofluorescence identification.(2)Construction of submandibular gland specific extracellular matrix hydrogel in vitro,using physical(lyophilized,mechanical)Concussion)and chemical(Triton X-100 surfactant)combined method to prepare submandibular gland extracellular matrix,and then mixed with heparin-hyaluronic acid hydrogel.(3)Experimental group: the first group is a simple submandibular gland cell group(Control group),the second group is submandibular gland cells and submandibular gland specific extracellular matrix;the third group: submandibular gland cells and hydrogel;the fourth group: submandibular gland cells and heparin-hyaluronic-extracellular matrix hydrogel group;at different time points,the morphology and functional expression of cells in each group after culture(cell viability,glycoprotein mucopolysaccharide secretion and related gene expression changes)to be detected.(4)The experimental data obtained is used for statistical analysis of relevant experimental results through SPSS 19.0 software.Results:(1)The immunofluorescence results of the isolated primary submandibular gland cells showed that the expression levels of the marker proteins AQP5,?-amylase and CK7 in the submandibular gland cells were greater than 90%,which was a positive result,proving that the cells were submandibular gland epithelial cells.(2)Morphological analysis of submandibular gland cells after cultured in HA-HP-ECM hydrogel: The growth of cells cultured in HA-HP-ECM hydrogel is spherical,and the cells will gradually increase as the culture time increases.Compared with the cells cultured in two-dimension,acinar and duct-like structures were found after 12 days of culture,suggesting that the construction of three-dimensional culture can make salivary gland cells form a multi-cell population with three-dimensional spatial structure in the microenvironment,and can proliferate and differentiate.(3)Live/Dead results showed that the number of living cells in the experimental group and the control group gradually increased.At the same detection time point,the number of viable cells in the HA-HP-ECM hydrogel cultured submandibular gland cells in the experimental group was more than that in the control group.(4)The results of q PCR showed that after three-dimensional culture of submandibular gland cells with HA-HP-ECM hydrogel,the m RNA expression of AQP5 and ?-amylase increased significantly at the two time points of 24 h and 48 h compared with the control group.The expression level of CK7 at 24 h was slightly lower than that of the control group,but by 48 h,its expression level was higher than that of the control group.It can be inferred that three-dimensional culture is more conducive to the biological functions of salivary gland cells.(5)The PAS staining results of cell glycogen showed that the PAS reaction positive substances in the control group were less stained.Although the amount of cells in the hydrogel and HA-HP-ECM hydrogel groups was small,all the cells in the results were dark purple positive,suggesting the experiment The group can secrete proteoglycan.Conclusion: The concentration of ECM(3%-20%)has a certain effect on the proliferation of submandibular gland cells.When the concentration is 5%,the cell survival rate is higher.After three-dimensional culture of submandibular gland cells with HA-HP-ECM hydrogel,spherical cell clusters can be observed,and genes related to the biological functions of salivary gland cells can be expressed.The secretory function of salivary glands can be exerted,which can provide certain experimental basis for the construction of salivary gland organoids. |
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