BACKGROUND:The cryopreservation technology enables tissues/cells to be stored for a long time in a low-temperature environment while maintaining the integrity of their activity and function,which is of great significance for the construction of cell therapy,tissue engineering and biological sample banks.Cryoprotective agents often contain dimethyl sulfoxide and serum.To avoid the toxic side effects of dimethyl sulfoxide,the complexity of serum components and immune responses,although some finished cryoprotective agents have been marketed,they are faced with many difficulties such as high cost and limited application.Therefore,there is an urgent need to develop a cryoprotective agent with clear components and the ability to solve the above problems.OBJECTIVE:To evaluate the effects of a novel cryoprotectant on cryopreservation efficiency of different tissue and cell sources.METHODS:By applying the novel cryoprotectant as an experimental group with the commercially available and widely used cryoprotectant(control group)to umbilical cord Wharton's jelly tissue,umbilical cord mesenchymal stem cells,umbilical cord blood/peripheral blood mononuclear cells,NK and CIK cells,comparative analyses were conducted in terms of cell morphology,number,viability,surface markers,differentiation potential,and cell-killing toxicity assay before cryopreservation and after resuscitation thawing.We confirmed the cryopreservation effect of the new cryoprotectant and its potential application value.RESULTS AND CONCLUSION:(1)The novel cryoprotectant facilitated the normal growth of cryopreserved Wharton's jelly tissue upon recovery,exhibiting mesenchymal stem cell morphology.No significant differences were observed between the experimental and control groups in terms of cell recovery rate,surface markers,and differentiation potential.(2)There was no significant difference in the number and viability of cells between the experimental group and the control group after cryopreservation of cord blood/peripheral blood mononuclear cells,and the cryo-resuscitated cell numbers and viability of derived NK cells/CIK cells did not show significant difference between the experimental and control groups.(3)For NK cells derived and differentiated from cord blood/peripheral blood mononuclear cells,there was no significant difference in the proportion of CD56+CD16+cell subpopulations between the experimental group and the control group.For CIK cells derived and differentiated from cord blood/peripheral blood mononuclear cells,there was no significant difference in the proportions of CD3+CD8+and CD3+CD56+cell subpopulations between the experimental group and the control group.(4)In terms of cytotoxicity testing,when the effective-target ratio of immune cells and melanoma cell line Mel624 was 20:1,whether it was NK cells/CIK cells derived from cord blood or peripheral blood mononuclear cells,there was no significant difference in the tumoricidal activity of cells between the experimental group and the control group.These findings suggest that the novel cryoprotectant can replace existing commercially available and widely used cryoprotectants,and is applicable to Wharton's jelly tissue,umbilical cord mesenchymal stem cells,umbilical cord blood/peripheral blood mononuclear cells,as well as NK and CIK cells,providing a solid technical foundation for the scaling,standardization,and commercialization of universal cryoprotectants.

Objective:To explore the value of lymphocyte subsets in predicting the efficacy and prognosis of non-small cell lung cancer(NSCLC)patients receiving programmed death receptor 1(PD-1)monoclonal antibody(mAb)combined with chemotherapy.Methods:A retrospective analysis was conducted on the clinical data of 50 NSCLC patients diagnosed and treated with PD-1 mAb combined with chemotherapy at the Second Hospital of Lanzhou University from January 2022 to December 2023.Peripheral blood lymphocyte subsets(including total T cells,CD4+T cells,CD8+T cells,NK cells,total B lymphocytes,and CD4+/CD8+T cell ratio)were collected before and after two cycles of treatment.After two cycles of treatment,imaging examination was performed to evaluate the therapeutic efficacy,dividing patients into disease control(DC)group and disease progression(PD)group.The relationship between lymphocyte subset levels and the short-term efficacy in NSCLC patients was analyzed using chi-square test,rank sum test,and Logistic regression analysis.The value of lymphocyte subsets in predicting patients'progression-free survival(PFS)was explored using Kaplan-Meier method.Results:PD-1 mAb combined with chemotherapy significantly influenced the immune status of NSCLC patients.After treatment,the peripheral blood CD4+T cells and CD4+/CD8+T cell ratio significantly increased(both P<0.01),while the level of CD8+T cells decreased.In terms of short-term efficacy,the proportion of CD4+T cells and the CD4+/CD8+T cell ratio in the DC group were significantly higher than those in the PD group(both P<0.01).Logistic multivariate analysis showed that the CD4+/CD8+T cell ratio was an independent factor affecting the efficacy of PD-1 mAb combined with chemotherapy.ROC curve analysis showed that the area under the curve(AUC)for CD4?/CD8? T cell ratio variation was 0.820(>0.5),with a cut-off value of 0.15.Patients with a the CD4+/CD8+T cell ratio increase of≥0.15 had a longer PFS.Conclusion:The proportion of CD4+T cells,CD8+T cells,and the CD4+/CD8+T cell ratio in peripheral blood can predict the efficacy and prognosis of PD-1 mAb combined with chemotherapy in advanced NSCLC patients.

Objective The purpose of this study is to utilize CiteSpace visualization software for literature analysis to investigate published literature concerning geriatric pharmacy outpatient services,create a knowledge graph,and analyze and discuss the developmental process and relevant hotspots of geriatric pharmacy outpatient services in China.Methods Utilizing the China National Knowledge Infrastructure(CNKI)as the source database,a search for literature containing the core phrases"pharmacy outpatient",'pharmacist outpatient","drug outpatient","drug consultation outpatient"and"elderly"was conducted from the inception of the database to March 2024 for data extraction.CiteSpace was employed to generate a visual knowledge graph,analyze,and discuss key nodes such as authors,institutions,and keywords within the realm of geriatric pharmacy outpatient services.Results After screening based on inclusion criteria,a total of 396 articles were included in the study.Scholars such as Ge Weihong,Wu Qiuhui and Liu Yang have made significant contributions to the research field of geriatric pharmacy outpatient services.Drum Tower Hospital affiliated with Nanjing University Medical School,the Sixth People's Hospital affiliated with Shanghai Jiao tong University,Dali University rank as the top three publishing institutions in terms of publication volume.The most frequently occurring keywords in the published literature include"pharmaceutical services","clinical pharmacists","pharmacy clinics","rational drug use"and"pharmacists".Conclusion"Drug safety","survey questionnaires"and"tertiary hospitals"have emerged as prominent and evolving topics within the realm of geriatric pharmacy outpatient services in China in recent years.In general,there remains considerable potential for further development in both the scope and depth of outpatient services for the elderly in the field of pharmacy in China.

BACKGROUND:The cryopreservation technology enables tissues/cells to be stored for a long time in a low-temperature environment while maintaining the integrity of their activity and function,which is of great significance for the construction of cell therapy,tissue engineering and biological sample banks.Cryoprotective agents often contain dimethyl sulfoxide and serum.To avoid the toxic side effects of dimethyl sulfoxide,the complexity of serum components and immune responses,although some finished cryoprotective agents have been marketed,they are faced with many difficulties such as high cost and limited application.Therefore,there is an urgent need to develop a cryoprotective agent with clear components and the ability to solve the above problems.OBJECTIVE:To evaluate the effects of a novel cryoprotectant on cryopreservation efficiency of different tissue and cell sources.METHODS:By applying the novel cryoprotectant as an experimental group with the commercially available and widely used cryoprotectant(control group)to umbilical cord Wharton's jelly tissue,umbilical cord mesenchymal stem cells,umbilical cord blood/peripheral blood mononuclear cells,NK and CIK cells,comparative analyses were conducted in terms of cell morphology,number,viability,surface markers,differentiation potential,and cell-killing toxicity assay before cryopreservation and after resuscitation thawing.We confirmed the cryopreservation effect of the new cryoprotectant and its potential application value.RESULTS AND CONCLUSION:(1)The novel cryoprotectant facilitated the normal growth of cryopreserved Wharton's jelly tissue upon recovery,exhibiting mesenchymal stem cell morphology.No significant differences were observed between the experimental and control groups in terms of cell recovery rate,surface markers,and differentiation potential.(2)There was no significant difference in the number and viability of cells between the experimental group and the control group after cryopreservation of cord blood/peripheral blood mononuclear cells,and the cryo-resuscitated cell numbers and viability of derived NK cells/CIK cells did not show significant difference between the experimental and control groups.(3)For NK cells derived and differentiated from cord blood/peripheral blood mononuclear cells,there was no significant difference in the proportion of CD56+CD16+cell subpopulations between the experimental group and the control group.For CIK cells derived and differentiated from cord blood/peripheral blood mononuclear cells,there was no significant difference in the proportions of CD3+CD8+and CD3+CD56+cell subpopulations between the experimental group and the control group.(4)In terms of cytotoxicity testing,when the effective-target ratio of immune cells and melanoma cell line Mel624 was 20:1,whether it was NK cells/CIK cells derived from cord blood or peripheral blood mononuclear cells,there was no significant difference in the tumoricidal activity of cells between the experimental group and the control group.These findings suggest that the novel cryoprotectant can replace existing commercially available and widely used cryoprotectants,and is applicable to Wharton's jelly tissue,umbilical cord mesenchymal stem cells,umbilical cord blood/peripheral blood mononuclear cells,as well as NK and CIK cells,providing a solid technical foundation for the scaling,standardization,and commercialization of universal cryoprotectants.

Objective The purpose of this study is to utilize CiteSpace visualization software for literature analysis to investigate published literature concerning geriatric pharmacy outpatient services,create a knowledge graph,and analyze and discuss the developmental process and relevant hotspots of geriatric pharmacy outpatient services in China.Methods Utilizing the China National Knowledge Infrastructure(CNKI)as the source database,a search for literature containing the core phrases"pharmacy outpatient",'pharmacist outpatient","drug outpatient","drug consultation outpatient"and"elderly"was conducted from the inception of the database to March 2024 for data extraction.CiteSpace was employed to generate a visual knowledge graph,analyze,and discuss key nodes such as authors,institutions,and keywords within the realm of geriatric pharmacy outpatient services.Results After screening based on inclusion criteria,a total of 396 articles were included in the study.Scholars such as Ge Weihong,Wu Qiuhui and Liu Yang have made significant contributions to the research field of geriatric pharmacy outpatient services.Drum Tower Hospital affiliated with Nanjing University Medical School,the Sixth People's Hospital affiliated with Shanghai Jiao tong University,Dali University rank as the top three publishing institutions in terms of publication volume.The most frequently occurring keywords in the published literature include"pharmaceutical services","clinical pharmacists","pharmacy clinics","rational drug use"and"pharmacists".Conclusion"Drug safety","survey questionnaires"and"tertiary hospitals"have emerged as prominent and evolving topics within the realm of geriatric pharmacy outpatient services in China in recent years.In general,there remains considerable potential for further development in both the scope and depth of outpatient services for the elderly in the field of pharmacy in China.

Objective To evaluate the effect of miRNA-29 (miR-29) family on the synthesis of collagen Ⅰ and Ⅲ in chronically photodamaged (photoaged) skin.Methods Some cultured human dermal fibroblasts (HDFs) were divided into 2 groups:non-irradiated group receiving no treatment,and chronic photodamage group treated with repetitive ultraviolet A (UVA) radiation,which served as a chronically photodamaged cell model and was verified by flow cytometry and β-galactosidase staining.Western blot analysis was performed to determine the protein expression of collagen Ⅰ and Ⅲ,and real-time fluorescence-based quantitative PCR (qRT-PCR) to measure expression of 3 members of the miR-29 family (miR-29a-3p,miR-29b-3p and miR-29c-3p) in the above 2 groups.The differentially expressed miR-29c-3p between the above 2 groups was chosen for further functional tests.Some HDFs were divided into 4 groups to be transfected with fluorescein-labelled miR-29c-3p mimics (overexpression group),inhibitors (inhibition group),and their control RNA oligonucleotides (negative control group and inhibitor control group) respectively.The transfection efficiency was evaluated by the proportion of fluorescent cells,and the relative expression of miR-29c-3p in the above 4 groups was measured by qRT-PCR for evaluating the RNA interference efficiency,qRT-PCR was conducted to determine the mRNA expression of collagen type Ⅰ α1 (COL1A1) and collagen type Ⅲ α1 (COL3A1) genes,and Western blot analysis to measure the protein expression of collagen Ⅰ and Ⅲ.Results Compared with the non-irradiated group,the chronic photodamage group showed significantly increased proportion of senescent cells (36.47％ ± 3.20％ vs.12.56％ ± 1.46％,P ＜ 0.01) and G1-phase cells (71.70％ ± 2.43％ vs.41.89％ ± 1.86％,P ＜ 0.01),but significantly decreased proportion of S-phase cells (10.63％ ± 0.36％ vs.36.48％ ± 1.31％,P ＜ 0.01),which indicated that the chronically photodamaged cell model was established successfully.The protein expression of collagen Ⅰ and Ⅲ was significantly lower in the chronic photodamage group (0.40 ± 0.19 and 0.52 ± 0.10) than in the non-irradiated group (1.00 ± 0.12 and 1.00 ± 0.10,respectively,both P ＜ 0.01).The expression of miR-29c-3p was significantly higher in the chronic photodamage group than in the non-irradiated group (4.42 ± 2.05 vs.0.89± 0.10,P ＜ 0.05),while there were no significant differences in the expression of miR-29a-3p or miR-29b-3p between the 2 groups (both P ＞ 0.05).Twenty-four hours after transfection,the overexpression group and inhibition group both showed more than 90％ transfection efficiency which met the interference requirements.The expression of miR-29c-3p was significantly higher in the overexpression group than in the negative control group (224.17 ± 2.00 vs.2.45 ± 0.34,P ＜ 0.01),but significantly lower in the inhibition group than in the inhibitor control group (0.20 ± 0.08 vs.2.24± 0.14,P ＜ 0.01),suggesting that a RNA interference model was successfully established.The mRNA expression of COL1A1 and COL3A1 and the protein expression of collagen Ⅰ and Ⅲ were significantly lower in the overexpression group than in the negative control group and inhibition group (all P ＜ 0.05),and significantly higher in the inhibition group than in the inhibitor control group (all P ＜ 0.01).Conclusion The expression of miR-29c-3p is up-regulated in chronically photodamaged HDFs,likely by regulating the mRNA expression of COL1A1 and COL3A1 and the protein expression of collagen Ⅰ and Ⅲ.

OBJECTIVEThis paper aims to study the effects of gossypol acetic acid (GAA) on proliferation and methylation level of human MutL homologue 1 (hMLH1) gene in human tongue cancer cell line Tca8113.

METHODSThe MTT assay was used to determine the effects of the acid on the proliferation inhibition in Tca8113 cells treated with different GAA concentrations. Nested methylation-specific polymerase chain reaction (nMSP) was used to detect the change in the methylation level of hMLH1 after 48 and 72 h with 30 and 15 micro mol L(-1) GAA treatment.

RESULTSMTT assay results showed the growth and proliferation inhibition of Tca8113 cells in the experimental GAA group after 24 h to 72 h of GAA treatment. The nMSP results indicated that the average optical density of hMLH1 in the Tca8113 cells significantly changed after the GAA treatment (30 micro mol L(-1) GAA for 48 h and 15 micro mol L(-1) for 72 h) (P<0.05) compared with that of the control group.

CONCLUSIONGAA does not only inhibit Tca8113 proliferation but also has a demethylation effect on the hMLH1 gene. These phenomena may be part of an underlying tumor-suppression mechanism of GAA.

Cell Line ; Cell Line, Tumor ; Cell Proliferation ; Gossypol ; analogs & derivatives ; Humans ; Methylation ; Tongue Neoplasms

Objective This paper aims to study the effects of gossypol acetic acid (GAA) on proliferation and methylation level of human MutL homologue 1 (hMLH1) gene in human tongue cancer cell line Tca8113. Methods The MTT assay was used to determine the effects of the acid on the proliferation inhibition in Tca8113 cells treated with different GAA concen-trations. Nested methylation-specific polymerase chain reaction (nMSP) was used to detect the change in the methylation level of hMLH1 after 48 and 72 h with 30 and 15μmol·L-1 GAA treatment. Results MTT assay results showed the growth and proliferation inhibition of Tca8113 cells in the experimental GAA group after 24 h to 72 h of GAA treatment. The nMSP results indicated that the average optical density of hMLH1 in the Tca8113 cells significantly changed after the GAA treat-ment (30μmol·L-1 GAA for 48 h and 15μmol·L-1 for 72 h) (P<0.05) compared with that of the control group. Conclusion GAA does not only inhibit Tca8113 proliferation but also has a demethylation effect on the hMLH1 gene. These phenomena may be part of an underlying tumor-suppression mechanism of GAA.