Objective:To investigate the composition of immune cells and fibroblastic reticular cells (FRCs) in the lymph nodes (LNs) follicles of human immunodeficiency virus (HIV)-infected individuals with varying immune statuses, and their association with HIV replication.Methods:Neck LNs samples were collected from 4 treatment-naive, newly diagnosed HIV-infected individuals with diverse immune statuses. RNA in situ sequencing was employed, with imaging achieved via rolling circle amplification and fluorescence labeling. By integrating cell segmentation and nuclear staining, single-cell data from up to one hundred thousand cells were generated per paraffin tissue section. Using lymphoid follicles as the unit of analysis, compositional changes in immune cells and FRCs were characterized, and their correlations with viral replication were evaluated. Results:The peripheral blood CD4 + T cell counts of samples LN_1, LN_2, LN_3, and LN_4 exhibited a sequential decrease. A total of 31, 15, 16, and 18 structurally intact follicles were identified in each sample, respectively. In the follicles of LN_1, the proportion of HIV-replicating cells positively correlated with cDCs abundance ( R2=0.2, P=0.011), and HIV RNA signals were spatially colocalized with cDCs and FRCs. In the follicles of LN_2, HIV RNA molecules showed preferential enrichment within FRCs. In sample LN_3, HIV RNA enrichment was observed in both cDCs and CD4 + T cells. In sample LN_4, the proportion of cells with HIV replication was positively correlated with the proportions of the following cells: cDCs ( R2=0.38, P=0.006 4), CD4 + T cells ( R2=0.28, P=0.025), and FRCs ( R2=0.26, P=0.029), and HIV RNA molecules were detected in cDCs, CD4 + T cells, and FRCs. LN_1 and LN_2 samples showed a trend toward negative correlation between HIV-replicating cell proportion and CD8 + T cells proportion. LN_4 sample demonstrated a significant positive correlation between HIV-replicating cell proportion and CD8 + T cells proportion ( R2=0.23, P=0.046). Conclusions:RNA in situ sequencing technology reveals unique distribution patterns of immune cells and viral replication in LNs follicles of HIV-infected individuals. The follicular immune microenvironment exhibits distinct characteristics associated with peripheral blood CD4 + T cell counts, providing novel insights into the spatial dynamics of HIV persistence and immune cell interactions during infection.

Objective:To investigate the composition of immune cells and fibroblastic reticular cells (FRCs) in the lymph nodes (LNs) follicles of human immunodeficiency virus (HIV)-infected individuals with varying immune statuses, and their association with HIV replication.Methods:Neck LNs samples were collected from 4 treatment-naive, newly diagnosed HIV-infected individuals with diverse immune statuses. RNA in situ sequencing was employed, with imaging achieved via rolling circle amplification and fluorescence labeling. By integrating cell segmentation and nuclear staining, single-cell data from up to one hundred thousand cells were generated per paraffin tissue section. Using lymphoid follicles as the unit of analysis, compositional changes in immune cells and FRCs were characterized, and their correlations with viral replication were evaluated. Results:The peripheral blood CD4 + T cell counts of samples LN_1, LN_2, LN_3, and LN_4 exhibited a sequential decrease. A total of 31, 15, 16, and 18 structurally intact follicles were identified in each sample, respectively. In the follicles of LN_1, the proportion of HIV-replicating cells positively correlated with cDCs abundance ( R2=0.2, P=0.011), and HIV RNA signals were spatially colocalized with cDCs and FRCs. In the follicles of LN_2, HIV RNA molecules showed preferential enrichment within FRCs. In sample LN_3, HIV RNA enrichment was observed in both cDCs and CD4 + T cells. In sample LN_4, the proportion of cells with HIV replication was positively correlated with the proportions of the following cells: cDCs ( R2=0.38, P=0.006 4), CD4 + T cells ( R2=0.28, P=0.025), and FRCs ( R2=0.26, P=0.029), and HIV RNA molecules were detected in cDCs, CD4 + T cells, and FRCs. LN_1 and LN_2 samples showed a trend toward negative correlation between HIV-replicating cell proportion and CD8 + T cells proportion. LN_4 sample demonstrated a significant positive correlation between HIV-replicating cell proportion and CD8 + T cells proportion ( R2=0.23, P=0.046). Conclusions:RNA in situ sequencing technology reveals unique distribution patterns of immune cells and viral replication in LNs follicles of HIV-infected individuals. The follicular immune microenvironment exhibits distinct characteristics associated with peripheral blood CD4 + T cell counts, providing novel insights into the spatial dynamics of HIV persistence and immune cell interactions during infection.

Objective To investigate the effect of nuclear factor of activated T cells 2(NFATc2)on the proliferation and apoptosis of human ovarian cancer cells and its possible mechanism.Methods The human epithelial ovarian cancer cell line A2780 was selected,and the A2780 cell lines with over expression(over ex-pression group)and low expression[transfection of small interfering RNA(siRNA),NFATc2 siRNA group]of NFATc2 were constructed,respectively.The control group without any treatment was established.Thiazole blue(MTT)and flow cytometry were used to detect the cell proliferation and apoptosis levels,respectively.The expression levels of apoptosis related proteins and CXC chemokine motif ligand 8(CXCL8)were detected by Western blot.Normal A2780 cells(the blank group),A2780 cells transfected with blank vectors(the con-trol group)and A2780 cells with low expression of NFATc2(the NFATc2 siRNA group)were used to estab-lish ovarian cancer model with subcutaneous xenograft.The tumor volume and mass were measured,and the expression level of CXCL8 in tumor tissue was detected by Western blot.Results Compared with the control group,the cell proliferation rate in the NFATc2 siRNA group was decreased,the cellular apoptosis rate was increased,the cleaved caspase-3 expression level was increased,the Bcl-2/Bax ratio was decreased,the CXCL8 expression was decreased,and the differences were statistically significant(P＜0.01).The cell lines in the o-ver expression group showed the opposite effect.The tumor mass and volume of nude mice,and the expression level of CXCL8 in the NFATc2 siRNA group were lower than those in the blank group and the control group(P＜0.01).Conclusion Down-regulation of NFATc2 could reduce the cell proliferation,increase the expres-sion of apoptosis related proteins by inhibiting CXCL8 expression in A2780 cells,thus promote the apoptosis of ovarian cancer cells.

OBJECTIVE To establish the fingerprints of Kangfuyan capsules and carry out chemical pattern recognition analysis，and simultaneously determine the contents of five components so as to promote the quality standard of the drug. METHODS High performance liquid chromatography （HPLC）fingerprints of 11 batches of Kangfuyan capsules （S1-S11）were established by Similarity Evaluation System of TCM Chromatographic Fingerprint （2012 edition）；identification and attribution analysis of chromatographic peaks were carried out by comparison with the chromatograms of the reference substance and the decoction pieces of single ingredient. SPSS 26.0 and SIMCA 14.1 software were used for cluster analysis and principal component analysis. HPLC method was used to determine the contents of matrine ，phellodendrine chloride ，rutin，forsythoside A and berberine hydrochloride. RESULTS There were 29 common peaks in the fingerprints for 11 batches of samples ，and the similarity was higher than 0.99. A total of 5 chromatographic peaks were identified ，which are matrine （peak 3），phellodendron chloride （peak 14），rutin （peak 20），forsythiaside A （peak 22） and berberine hydrochloride （peak 28）. The results of cluster analysis and principal component analysis showed that S 1-S9 were clustered into one category ，and S 10 and S 11 were clustered into another category. The contents of above 5 components were 29.320 5-60.144 3，0.621 6-1.076 6，1.025 9-2.830 5，2.899 3-6.212 7 and 4.425 1-8.581 6 mg/g， respectively. CONCLUSIONS The established fingerprint and content determination method are stable and reliable ，and can provide reference for the quality control of the preparation in combination with chemical pattern recognition analysis.

OBJECTIVE：To compare chemical composition types and ginsenoside content of Panax notoginseng flowers with different growing years ，and to explore the effect of growing year on the quality of P. notoginseng flowers. METHODS ：Each 10 batches of biennial，triennial and quadrennial P. notoginseng flower were collected and determined by HPLC. The determination was performed on Shim-pack GIST C 18 column with mobile phase consisted of acetonitrile- 0.05％ phosphoric acid solution （gradient elution ）at the flow rate of 0.5 mL/min. The column temperature was set at 30 ℃，and the detection wavelength was set at 203 nm. The sample size was 20 μL. Similarity Evaluation System of TCM Chromatogram Fingerprint was used to establish the fingerprint of 30 batches of samples ，identify the diagnostic components and analyze the similarity. Cluster analysis was conducted by using SPSS 22.0 software. The contents of ginsenoside Rb 1，Rb2，Rb3 and Rc in 30 batches of P. notoginseng flower with different growing years were determined by above HPLC . The quality control analysis was conducted by using SPSS 22.0 software. RESULTS：Established fingerprint showed good precision ，stability and reproducibility. There were good linear relationship （R2＞ 0.999），quantitative limit ，precision，stability，repeatability and accuracy of the content determination method . Six common components as ginsenoside Rb 1， Rb2， Rb3 and Rc were Δ 基金项目：云南省地方高校联合专项（No.KX182504Y） identified in P. notoginseng flower with different growing ＊助教，硕士。研究方向：中药资源开发 。电话：0876-2684947。 E-mail：wshuangzaiqiang@163.com years by fingerprint ；ginsenoside Rd was identified in triennial # 通信作者 ：研究员，硕士。研究方向 ：中药资源开发 。电话： P. notoginseng flower. The similarities of the fingerprints 0876-8883731。E-mail：gaomingju@163.com among 10 batches of biennial ，triennial and quadrennial P. 中国药房 2020年第31卷第8期 China Pharmacy 2020Vol. 31 No. 8 ·969· notoginseng flower were 0.881，0.952 and 0.945，respectively. The similarity among samples with different growing ye ars was more than 0.817. Thirty batches of P. notoginseng flower could be grouped into 4 categories，the category Ⅱ was quadrennial samples，the category Ⅲ was triennial samples ，while the categories Ⅰ and Ⅳ were mostly biennial samples and a small number of triennial and quadrennial samples. RSDs of 4 ginsenosides contents and their total contents in biennial samples were 8.90％-21.43％ and total saponin contents were 11.65％-17.76％，respectively. RSDs of 4 ginsenosides contents and their total contents in triennial samples were 6.45％-14.23％，and total saponin contents were 15.74％-19.30％. RSDs of 4 ginsenosides contents and their total contents in quadrennial samples were 7.50％-18.86％，and total saponin contents were 15.92％-20.16％. The results of quality control analysis showed that biennial samples mainly distributed in the areas of Ⅱ and Ⅲ ；triennial and quadrennial samples mainly distributed in the areas of Ⅰ and Ⅱ ；the order of ginsenosides content was Ⅰ ＞Ⅱ ＞Ⅲ. CONCLUSIONS：Chemical components of P. notoginseng flower with different growing years are generally close in types but there still a re some differences ，among which the content of ginsenosides in biennial samples is lower ，fluctuates more ，and the overall quality is slightly poor ；the content of ginsenosides in triennial and quadrennial samples is higher ，fluctuates less ，and the overall quality is higher and tends to be stable.

Objective To investigate the distribution of human papillomavirus(HPV)in different age groups and its correlation with the degree of cervical lesions.Methods 2 031 patients with HPV screening specimens in our hospital from May 2012 to May 2015 were chosen as study subjects.To acquire patient cervical squamous columnar epithelial cells at the junction to detect HPV DNA types.HPV -positive patients used ultra -thin liquid -based cervical cytology technology (TCT)to detect.Patients with abnormal TCT detection were performed with electronic biopsy,and the diagnosis was made by pathology biopsy,and analyzed the distribution of HPV in different age groups and different degree of cervical lesions.Results In different age groups,≤20 years of age groups of patients with high -risk HPV positive rate was 19.7%,which was significantly higher than that of >20 -30 years,>30 -40 years and >40 -50 years three groups (14.4%,13.9%,15.0%)(χ2 =4.259,5.724,3.988,all P <0.05).There was no significant difference with the 50 years old group (17.1% )(χ2 =1.724,P >0.05).There was no significant difference in HPV positive rate among the ≤20 years,>20 -30 years,>30 -40 years,>40 -50 years,>50 years ages patients with high risk HPV infection group and low risk HPV infection group(χ2 =0.679,1.021,0.968,0.736, 0.668,all P >0.05).In the high -risk HPV infected group,the risk of single type infection in >20 -30 years,>30 -40 years,>40 -50 years,>50 years ages patients were significant higher than the mixed types infection(χ2 =4.213,3.894,4.256,5.330,5.666,all P <0.05).Infected patients of all ages with high -risk HPV were HPV16, HPV52,HPV58 type -based,low -risk type HPV43 type places mainly.More than 31 to 40 years age group of patients infected with high -risk HPV types in cervical lesions mainly cervicitis and 40 years and older group of patients with high -risk HPV infection incidence of invasive cervical cancer were significantly higher.Conclusion Different age groups of women for HPV genotyping is important for diagnosis of cervical lesions.

Objective To explore biological effects of up-regulated expression of transfected FOLR1 gene on SKOV3 cell lines following action by cisplatin(DDP).Methods Three groups of cells originated from the same SKOV3 cell line were used in this research,including the SKOV3 cell line (blank control),the cell line transfected with lentiviral pWPI plasmid (no-load control) and the cell line transfected with FOLR1 gene via lentiviral pWPI plasmid (experimental group).Next,the mRNA and protein expression of FOLR1 gene in the three groups were detected by reverse transcription (RT)-PCR and western blot,respectively.Methyl thiazolyl tetrazolium (MTT) assay was used to analysis cells growth curve and identify their sensitivity to cisplatin,and their half inhibition concentration (IC5o) values were calculated.Based on the IC50 value (3.6 μg/ml) in the experimental group,different levels of cisplatin concentration (0.5 × IC50,1 × IC50,2 × IC50,respectively) were administered to the three groups of cells,and the inhibition rates,apoptosis rates as well as apoptosis proportion of each group after 24,48,72 hours were further recorded.Finally,the residual cisplatin concentrations in the three group cells acted successively by 1 × IC50 cisplatin for 48 hours were measured by high performance liquid chromatography(HPLC).P value less than 0.05 were defined as statistically significant.Results RT-PCR and western blot detection showed that stable mRNA and protein expression of the FOLR1 gene in the experimental group while the other two groups were not.MTT assay demonstrated that higher cell growth rate,sensitivity to cisplatin(IC50 =3.6 μg/ml) and inhibition rate in the experimental group compared with those in the other two groups (P ＜ 0.05),which showed no significance in intergroup comparison(P ＞ 0.05).Flow cytometry showed apoptosis rates among three groups increased with higher cisplatin concentrations and longer action duration in dosage-time dependent manner (P ＜ 0.05),and the proportion of S phase cells increased with higher cisplatin concentration in dosage-dependent manner (P ＜ 0.05) ; for the same concentration and duration,the experimental group showed significantly different apoptosis rates and S phase cells compared with the other two groups,which demonstrated no significance in intergroup comparison (P ＞ 0.05).After action by cisplatin(3.6 μg/ml) for 48 hours,HPLC showed significantly higher residual cisplatin concentration (2.60±0.21) μg/106 cell counts in experimental group than those in no-load control group (1.49 ±0.12) μg/106 cell counts and blank control group (1.54 ± 0.11)xg/106 cell counts,respectively (P ＜0.05),and the comparison within the latter two groups showed no significance (P ＞ 0.05).Conclusion Up-regulated expression of the transfected FOLR1 gene in SKOV3 cells may be associated with higher sensitivity to cisplatin,residual cisplatin concentration and higher proportion of S phase cells,and tended to inhibit cancer cells growth and induce apoptosis.

Through this research a lentiviral vector expressing the gene of folate-binding protein-1 (FOLR1) was constructed and the corrsponding expression products were identified. Firstly, full-length of the FORL1 gene was amplified by PCR and cloned into the plasmid pWPI. Then it was further confirmed by PCR and sequencing. Secondly, after the recombinant pWPI and its helper plasmid co-transfected the virus packaging 293T cells, SKOV3 cells were infected with the virus particles and sorted by flow cytometry. Thirdly, the FOLR1 gene was detected by RT-PCR and its protein expression was detected by Western blot. Finally, the recombinant expression vector was successfully constructed, and lentiviruses were successfully packaged by the 293T cells. A great quantity of green fluorescent cells could be seen after the SKOV3 cells were effectively infected with the lentiviruses carrying the FOLR1 gene. The sorting could be done and detected by cytometrying the FORL1 gene and its stable expression by the two methods above, which laid experimental foundation for exploring its biological function in ovarian cancers.
Cell Line ; Cell Line, Tumor ; Cloning, Molecular ; Female ; Folate Receptor 1 ; biosynthesis ; genetics ; Genetic Vectors ; genetics ; Green Fluorescent Proteins ; biosynthesis ; genetics ; Humans ; Kidney ; cytology ; Lentivirus ; genetics ; metabolism ; Ovarian Neoplasms ; pathology ; Polymerase Chain Reaction ; Recombinant Proteins ; biosynthesis ; genetics ; Transfection

60 years old). 43 cases of antiepileptic drugs combination accounted for 10% and the therapeutic plasma concentration of 27 cases deviated from normal range(62.8%). CONCLUSION:Results of plasma concentration monitoring provide an important basis for clinical drug use. Monitoring data and other clinical index can promote rational use of antiepileptic drugs.