Objective:To establish an in vitro culture system of small intestinal organoid in normal mice and perform functional identification, and to provide an in vitro research tool for material transport in the intestine under chronic kidney disease. Methods:The small intestinal crypts of C57BL/6J mice were isolated, extracted and cultured in an in vitro three dimension culture system. The formation of small intestinal organoid was observed with inverted microscope. The tissue structure of the small intestinal organoid was observed by hematoxylin and eosin staining. The cellular composition of the small intestinal organoid was identified by immunofluorescence. The expression of substance absorption-related transporters in the small intestinal organoid was detected by real time fluorescence quantitative PCR. Results:The small intestinal crypts were successfully extracted. The organoids of small intestine and different intestinal segments were successfully constructed. The cultured organoids had vigorous proliferation ability and maintained proliferation ability after passing through generations. Immunofluorescence results showed that the small intestinal organoids expressed mucin2, chromogranin A, oflm4 and lysozyme, which were different types of intestinal cell biomarkers. The PCR results showed that small intestinal organoids expressed calcium, phosphate and sodium absorption-related transporters, and the mRNA expression levels of major transporters for sodium and phosphate absorption in different intestinal segments-like organs were consistent with those in vivo, which was consistent with the characteristics of small intestinal segmental absorption. Conclusions:The successful construction of small intestine and different intestinal segments organoids, and the first observation of the expression of substance absorption-related transporters in such organoid, provide a stable and convenient in vitro research tool for the development of intestinal substance transport in chronic kidney disease.

In order to investigate the molecular mechanism of silk/threonine protein kinase (STK)-mediated blue light response in the algal Chlamydomonas reinhardtii, phenotype identification and transcriptome analysis were conducted for C. reinhardtii STK mutant strain crstk11 (with an AphvIII box reverse insertion in stk11 gene coding region) under blue light stress. Phenotypic examination showed that under normal light (white light), there was a slight difference in growth and pigment contents between the wild-type strain CC5325 and the mutant strain crstk11. Blue light inhibited the growth and chlorophyll synthesis in crstk11 cells, but significantly promoted the accumulation of carotenoids in crstk11. Transcriptome analysis showed that 860 differential expression genes (DEG) (559 up-regulated and 301 down-regulated) were detected in mutant (STK4) vs. wild type (WT4) upon treatment under high intensity blue light for 4 days. After being treated under high intensity blue light for 8 days, a total of 1 088 DEGs (468 upregulated and 620 downregulated) were obtained in STK8 vs. WT8. KEGG enrichment analysis revealed that compared to CC5325, the crstk11 blue light responsive genes were mainly involved in catalytic activity of intracellular photosynthesis, carbon metabolism, and pigment synthesis. Among them, upregulated genes included psaA, psaB, and psaC, psbA, psbB, psbC, psbD, psbH, and L, petA, petB, and petD, as well as genes encoding ATP synthase α, β and c subunits. Downregulated genes included petF and petJ. The present study uncovered that the protein kinase CrSTK11 of C. reinhardtii may participate in the blue light response of algal cells by mediating photosynthesis as well as pigment and carbon metabolism, providing new knowledge for in-depth analysis of the mechanism of light stress resistance in the algae.
Chlamydomonas reinhardtii/genetics* ; Photosynthesis/genetics* ; Plants/metabolism* ; Protein Kinases ; Threonine/metabolism* ; Carbon/metabolism* ; Serine/metabolism*