BACKGROUND:Stem cell transplantation provides a new approach to treat chronic renal disease.Specific marking and in vivo tracing of stern cells are the basis of studies in this field.However,the marking methods appropriate for all cells remain uncertain.OBJECTIVE:To observe the in vivo location and differentiation of 4',6-diamidino-2-phenylindole (DAPI) and green fluorescence protein (GFP)-Iabeled cells in adriamycin nephrosis rats so as to explore an efficient labeling and tracing method for metanephric mesenchymal cells (MMCs) derived from embryonic rats.DESIGN,TIME AND SETTING:Grouping comparative observation was performed at the Second Xiangya Hospital of Central South University from April to December 2007.MATERIALS:A total of 60 female SD rats,weighing 180-220 g,of dean grade,were used to establish models of adriamycin nephrosis.METHODS:DAPI and MMCs infected with GFP and DAPI were respectively injected into addamycin nephrosis via the tail vein.DAPI and GFP distribution in the frozen sections was detected at 1,3,and 5 weeks,postoperatively.In addition,GFP expression in renal tissues was detected by ABC immunoenzymatic staining method.MAIN OUTCOME MEASURES:DAPI and GFP-labeled Cell grafts in adriamycin nephrosis rats were compared.The changes of GFP-transfected MMCs at different time points were observed.RESULTS:DAPI positive cells were observed in tubular structures after 1 weeks of injection of DAPI-labeled cells and DAPI alone,and remained existing at 5 weeks,but the florescence was reduced with time.GFP-transfected MMCs were able to survive and integrate into tubular structures after 1 week,and remained existing at 5 weeks.Moreover,the fluorescence was not reduced.ABC immunoanzymatic staining showed that only a few GFP-positive MMCs appeared in glomerular tufts,and mainly distributed in cytoplasm.Semi-quantitative evaluation of GFP show that the positive cell rate in rats with early application was greater than that with advanced application,and the positive rate was increased with time.CONCLUSION:Liposome mediated GFP gene transfer was an efficient labeling in vitro and suitable tracing method for cell differentiation experiment in vivo,suitable for short-term tracing and observation of transplanted cells.

Objecfive To detect the functional repair of metanephric mesenchymal cells (MMCs) transplantation in adriamycin (ADR)-induced glomerulopathy rats. Methods A total of 90 Sprague-Dawley female rats were randomly divided into three groups:ADR group (n=40,rats were injected via the tail vein with O.25 mg ADR/100 g body weight on days 1 and 21),ADR- MMCs group(n=40,rats were injected via the tail vein with 5×10~6-7×10~6 MMCs 8 weeks after the second ADR administration),control(n=10).All the rats were scarified 8 weeks after MMCsinjection.Pathology and collagen IV expression in renal tissue were examined.Moreover,matrix metalloproteinases 2 (MMP-2) and matrix metallopmteinases 9 (MMP-9) expression in the renal tissue were also detected with immunohistochemistry,and quantity analysis of protein and gene was further demonstrated with Westem blot and RT-PCR analysis,respectively. Results There were no significant differences in tubulointerstitial injury score and glomerulosclerosis degree between ADR group and ADR-MMCs group(P>0.05).Compared with ADR group,collagen Ⅳ and MMP-2 expression decreased, MMP-9 expression incrased in renal tissue of ADR-MMCs group, and the difference was significant (P<0.05). Conclusion MMCs transplantation may have potentially therapeutic effect on renal tissue fibrosis of adriamyein-induced glomerulopathy in rats, and the signaling pathways of MMPs appear to be involved in these processes.

OBJECTIVE：To study the effect of total flavonoids of Epimedium brevicornu on postmenopausal osteoporosis （PMOP）model rats based on BMP/Runx 2/Osx signaling pathway so as to confirm the mechanism of preventing and treating osteoporosis（OP）. METHODS ：By body mass stratification ，50 rats were randomly divided into sham operation group ，model group，E. brevicornu total flavonoids low-dose and high-dose groups [265，530 mg/（kg·d）]，estradiol group [0.09 mg/（kg·d）]， with 10 rats in each group. Except that sham operation group underwent sham operation ，PMOP model was established by ovariectomy and castration in other groups. After modeling ，they were given normal diet for 2 months and then given relevant medicine intragastrically for consecutive 84 d，once a day ；rats in sham operation group and model group were given equal volume of normal saline. After last medication ，bone mineral density （BMD）of femur and vertebrae of the right lower limb ，the number of trabecular bone （Tb.N），trabecular bone thickness （Tb.Th）and trabecular separation （Tb.Sp）of femur were determined in each group. The serum levels of Ca 2+，OC and P 1NP in serum were detected by ELISA. HE staining was used to observe pathological changes of femur. mRNA and protein expressions of BMP ，Runx2 and OSX in bone tissue were detected by RT-PCR and Western blotting. RESULTS ：Compared with sham operation group ，BMD of femur and vertebrae ，serum levels of Ca 2 +，OC and P 1NP， Tb.N and Tb.Th of femur ，mRNA and protein expression of BMP ，Runx2 and Osx in femur were decreased significantly in model group，while Tb.Sp of femur was increased significantly （P＜0.01）；the structure of trabecular bone was disordered and the fracture was obvious. Compared with model group ，above indexes of rats in administration groups were improved significantly （P＜ 0.05 or P＜0.01），and the effect of E. brevicornu total flavonoids was dose-dependent （P＜0.05）；the number of trabecular bone increased， arranged orderly and the structure was more brevicornu total flavonoids can improve OP ，the mechanism of which may be associated with com promoting the activity of BMP/Runx 2/Osx signaling pathway.

OBJECTIVE: To develop a genotyping method for the Junior blood type and report on a rare blood type with Jr(a-). METHODS: Healthy O-type RhD+ volunteer donors of the Shenzhen Blood Center from January to May 2021 (n = 1 568) and a pedigree with difficult cross-matching (n = 3) were selected as the study subjects. Serological methods were used for proband's blood type identification, unexpected antibody identification, and antibody titer determination. Polymerase chain reaction-sequence specific primer (PCR-SSP) method was used for typing the proband's RhD gene. ABCG2 gene coding region sequencing and a PCR-SSP genotyping method were established for determining the genotypes of the proband and his family members and screening of Jra antigen-negative rare blood type among the 1 568 blood donors. RESULTS: The proband's ABO and RhD blood types were respectively determined as B and partial D (RHDDVI.3/RHD01N.01), Junior blood type Jra antigen was negative, and plasma had contained anti-D and anti-Jra. Sequencing of the ABCG2 gene revealed that the proband's genotype was ABGG201N.01/ABGG201N.01 [homozygous c.376C>T (p.Gln126X) variants], which is the most common Jr(a-) blood type allele in the Asian population. Screening of the voluntary blood donors has detected no Jr(a-) rare blood type. Statistical analysis of the heterozygotes suggested that the allelic frequency for ABCG2*01N.01 (c.376T) was 0.45%, and the frequency of Jr(a-) rare blood type with this molecular background was about 0.2‰. CONCLUSION A very rare case of partial DVI.3 type and Jr(a-) rare blood type has been identified. And a method for identifying the Junior blood type through sequencing the coding regions of the ABCG2 gene and PCR-SSP has been established.
Humans ; Blood Group Antigens/genetics* ; Genotype ; Genotyping Techniques ; Heterozygote ; Alleles ; Blood Donors ; Rh-Hr Blood-Group System/genetics*

OBJECTIVE：To study the mechanism of H uanglian jiedu decoction （HJD）regulating macrophage polarization in order to explore its anti-inflammatory mechanism. METHODS ：The active components and predicted targets of HJD were screened through TCMSP and Swiss Target Prediction database ；the related targets of macrophage polarization were obtained by GeneCards and OMIM database ，and the network diagram of active ingredient-macrophage polarization target of HJD was drawn by using Cytoscape 3.6.0 software；protein interaction network was constructed by String database and core targets were extracted. Gene ontology（GO）enrichment analysis and Kyoto Encyclopedia of genes and genomes （KEGG）pathway enrichment analysis were carried out by using Cytoscape 3.6.0 software and DAVID website. Combined with the results of network pharmacology analysis ， RAW264.7 macrophage cells were divided into blank control group ，model group ，simvastatin group （10 μmol/L）and serum containing HJD group （obtained from the blood after the rats were given HJD at the dose of 10 g/kg）. Except the blank control group and model group were added culture medium ， the other groups were added with 100 μ L of relevant drug solution or serum containing drug. After 2 h of culture ，except for the blank control group ，LPS solution （100 μg/L）was added to the other groups for 24 h to induce inflammation. Western blot assay was used to detect the expression of AMPK. mRNA expression of M 1 type polarization factor （IL-1 β，iNOS）and M 2 type polarization factor （IL-10，Fizz1）were detected by RT-PCR. RESULTS ：A total of 50 active components of HJD （such as acacetin ，wogonin，quercetin，β-sitosterol）were screened ， which could regulate macrophage polarization through 12 GO items （such as anoikis ，astrocyte activation ），and 20 KEGG pathways（such as estrogen signaling pathway ，bladder cancer pathway ，AMPK signaling pathway ）. The results of cell test showed that compared with blank control group ，the expression of AMPK protein ，Fizz1 and IL- 10 mRNA in model group were significantly decreased （P＜0.01），while the expression of IL- 1β and iNOS mRNA were significantly increased（P＜0.01）； compared with model group ，the expression of AMPK protein ，Fizz1 and IL- 10 mRNA in serum containing HJD group and simvastatin group were significantly increased （P＜0.05 or P＜0.01），while the expression of IL- 1β and iNOS mRNA were significantly decreased （P＜0.01）. CONCLUSIONS ：HJD can regulate macrophage polarization through multiple targets and pathways；it can up regulate the expression of M 2-type polarization factors and down-regulate the expression of M 1-type polarization factors through AMPK signaling pathway ，regulate macrophage polarization and play an anti-inflammatory role.