ObjectiveTo study the effect of tumor associated neutrophil (TAN) releasing a proliferation-inducing ligand (APRIL) on the proliferation of pancreatic cancer cells in microenvironment.Methods① The expressions of APRIL in neutrophils (differentiated by HL-60 cell) and TAN cells were detected by use ELISA. ② The expressions of APRIL receptors B cell maturation antigen (BCMA) and trans-membrane activator and CAML interactor (TACI) in pancreatic cancer cell line PANC-1 were confirmed by use Western blotting. ③ Pancreatic cancer PANC-1 cells were co-cultured with TAN, and divided into a PANC-1 control group (referred to as the control group), a PANC-1+TAN treatment group (referred to as the PANC-1+TAN group), PANC-1+TAN+APRIL antibody treatment group (referred to as PANC-1+TAN+APRIL group), and PANC-1+rtificial recombinant APRIL protein (rAPRIL) treatment group (referred to as PANC-1+rAPRIL group). The CCK8 method was used to determine TAN release of APRIL on PANC-1 effect of cell proliferation activity.Results① The APRIL content in the culture medium of TAN cell group was higher than that of neutrophil group [(556.20±84.38) pg/mL vs. (377.17±57.07) pg/mL, P=0.038]. ② PANC-1 cells express the receptors BCMA and TACI of APRIL. ③ PANC-1 cell activity of PANC-1+TAN group and PANC-1+rAPRIL group [(126.80±1.42)%, (168.95±12.54)%] were significantly higher than the control group [(100 ± 0.00)%, P<0.05, P<0.001], the activity of PANC-1 cells in the PANC-1+TAN group was significantly higher than that in the PANC-1+TAN+APRIL group [(86.29 ± 12.20)%, P=0.003] and significantly lower than that of PANC-1+rAPRIL group (P=0.002), the activity of PANC-1 cells in PANC-1+rAPRIL group was significantly higher than that in PANC-1+TAN+APRIL antibody group (P<0.001).ConclusionIn the microenvironment of pancreatic cancer, the release of APRIL from TAN increases, which promotes the proliferative activity of PANC-1 in pancreatic cancer cells, which provides a new idea for the mechanism research and treatment of pancreatic cancer progression.
Objective To investigate the effect of bone morphogenetic protein 2 (BMP-2) and dexamethason (DXM) on proliferation and differentiation of human dental pulp cellsin vitro. Methods Primary human dental pulp cells were cultured in vitro by tissue culture method. The 3rd generation cells were used to identify cell phenotype for vimentin and cytokeratin by immunocytochemistry staining. The 3-5 generations of human dental pulp cells were randomly divided into 4 groups: 100 ng/mL BMP-2 (group A), 1×10–8 mol/L DXM (group B), and both 100 ng/mL BMP-2 and 1×10–8 mol/L DXM (group C) were added; neither BMP-2 nor DXM was added in group D as control group. The cell growth curve was drawn at 1, 3, 5, and 7 days after culture. The expressions of osteo/dentanogenic genes including alkaline phosphatase (ALP), dentin sialophoshoprotein (DSPP), and dentin matrix protein 1 (DMP-1) were detected by RT-PCR analysis at 5 and 7 days after culture, the ratio between the positive staining area and the total area by ALP staining at 14 days, and absorbance (A) value at 562 nm by alizarin red staining at 21 days after culture. Results Human dental pulp cells were successfully isolated and cultured, which were long fusiform and showed a positive reaction for vimentin and a negative reaction for cytokeratin. The growth curve indicated that cells increased with the extending of incubation time, reached a peak at 5 days, then reduced at 7 days to the level at 3 days. At 5 days after culture, the cells were significantly more in groups A, B, and C than group D (P<0.05), in group C than group A (P<0.05), and in group A than group B (P<0.05). RT-PCR analysis showed that the mRNA expressions of ALP, DSPP, and DMP-1 at 5 days were significantly higher in groups A, B, and C than group D (P<0.05), and in group C than groups A and B (P<0.05), but no significant difference was found between groups A and B (P>0.05); the mRNA expression of DSPP in groups A, B, and C was significantly higher than that in group D (P<0.05), but there was no significant difference in mRNA expressions between other groups at 7 days (P>0.05). At 14 days, positive staining in varying degrees was observed in each group, especially in group C; the ratio between the positive staining area and the total area was significantly higher in group C than groups A, B, and D (P<0.05), and in groups A and B than group D (P<0.05), but there was no significant difference between groups A and B (P>0.05). At 21 days, there were a variety of mineralized nodules in groups A, B, and C in nonuniformly scattered or clustered distribution, but no mineralized nodules were observed in group D. TheA values of mineralized nodules showed significant difference between groups (P<0.05). Conclusion BMP-2 may be more effective in promoting proliferation of human dental pulp cells than DXM. Combined application of BMP-2 and DXM can remarkably promote the proliferation and differentiation of human dental pulp cells.
ObjectiveTo observe apoptosis and proliferation of choledochus wall epithelial cell and fibrocyte, to understand the effects of apoptosis and proliferation on choledochal cyst development.MethodsThirty two cases of cystic dilatation,35 cases of cylindrical dilatation，and 25 cases of cholangiectasis caused by choledocholith were collected. All specimens were offered by department of hepatobiliarypediatric surgery. The apoptosis related index (bcl2 and bax) and cell proliferation index (PCNA) were detected by the immunohistochemical technique; Apoptosis was detected by TUNEL method. ResultsThere was serious mucosal epithelial cell damage in cystic dilatation group. In cylindrical dilatation group there was a damage similar to that of the cystis dilatation group, but the damage was not serious. In control group there was little damage in the duct wall, but there was a low positive rate of apoptosis of 〔epithelium cell (2.74±1.00)% and fibroblast (2.95±0.87）%〕, and a low bcl2 and bax’s expression rate, and a high PCNA’s expression rate 〔epithelium cell (3.74±1.00)%, fibroblast (3.71±1.77)%〕. There was no obvious difference between cylindrical dilatation group and cystic dilatation group (Pgt;0.05): the PCNA’s expression rate was low 〔(0.99±0.51)% and (0.90±0.38)% respectively〕, the bax expression rate was high in remaining epithelial cell, and the positive rate of bax was apparently higher than that of bcl2 (P＜0.05), the positive rate of the apoptosis cell was high 〔(13.94±4.77)%, (7.51±3.46)%〕; the expression rate PCNA were high 〔(9.91±2.91)%,(9.70±3.18)%〕, and expression rate of bax’s was low in the fibre tissue, the positive rate of bcl2 was markedly higher than that of bax, and the positive rate of the apoptosis cell was low 〔(3.74±2.12)%,(4.46±2.41)%〕. There were no marked difference between the two groups (Pgt;0.05). The expression of bcl2 and bax had marked difference both in cylindrical dilatation group and cystic dilatation group and as compared to control group (P＜0.05). ConclusionApoptosis has certain promoting effect in the course of choledochal cyst formation.
摘要:目的:探索槐耳清膏對體結腸癌SW480細胞增殖能力影響及其機制。方法:采用噻唑藍(MTT)比色法檢測槐耳清膏對SW480細胞增殖能力的作用,并探求最佳作用濃度;將體外培養細胞隨機分為常氧組(NC組)、低氧組(HC組)和低氧槐耳組(HH組),逆轉錄聚合酶鏈反應（RTPCR）檢測各組血管內皮生長因子(VEGF) mRNA表達水平,Western blot檢測蛋白表達水平。結果:槐耳清膏對SW480細胞抑制率隨藥物濃度增加而上升,1 mg/mL時抑制率最大(66.7%),與氟尿嘧啶組(濃度為10 μg/mL)相比無統計學意義。HH組和HC組VEGF mRNA表達均顯著高于NC組,分別為4.71±0.07,4.54±0.02和1.19±0.03(P＜0.05),但HH組與HC組比較差異無統計學意義。HC組VEGF蛋白表達顯著高于NC組,分別為0.66±0.03和0.38±0.02(P＜0.05),HH組較HC組VEGF蛋白表達均顯著下降,分別為0.37±0.03和0.66±0.03(P＜0.05)。結論:槐耳清膏可抑制SW480細胞增殖,1 mg/mL時抑制率最大。其機制為槐耳清膏下調細胞內VEGF蛋白表達,從而抑制腫瘤生長。Abstract: Objective: To investigate the effect of Huaier cream on proliferation of colon cancer cells SW480 and its mechanism. Methods: The proliferation was analyzed by MTT. SW480 cells were randomly divided into normoxic group (NC group), hypoxia group (HC group) and hypoxia group treated by Huaier (HH group). Levels of mRNA and protein expression of VEGF were detected by RTPCR and Western blot, respectively． Results: Huaier cream induced a dosedependent inhibition of SW480 cells. The maximum percentage of growth inhibition was 66.7% at a concentration of 1.0 mg/mL, but no significant difference was found compared to the positive control (5FU 10 μg/mL). VEGF mRNA levels were significantly higher in HC group and HH group than in NC group (4.71±0.07, 4.54±0.02 vs 1.19±0.03, all Plt;0.05), but not significantly different between HC group and HH group. VEGF protein expression was higher in HC group than NC group (0.66±0.03 vs 0.38±0.02, Plt;0.05). In HH group, VEGF protein was inhibited remarkably compared with HC group (0.37±0.03 vs 0.66±0.03, Plt;0.05). Conclusion: Huaier cream can significantly inhibit SW480 cells and the top inhibition concentration is 1.0 mg/mL. Huaier cream plays a role in inhibiting tumor through downregulating protein expression of VEGF.
ObjectiveTo investigate the role of endoplasmic reticulum stress in liver regeneration after associating liver partition and portal vein ligation for staged hepatectomy (ALPPS).MethodsSeventy-two C57bl/6 mice were randomly divided into ALPPS group, portal vein ligation group (PVL group), and sham operation group (Sham group), 24 mice in each group. And then one-stage ALPPS operation, simple PVL, and sham operation will be performed. Six mice were randomized selected of the three groups on the 1st, 2nd, 4th, and 7th day after surgery, respectively, the liver weight to body weight ratio (FLR/BW) of each group was measured, and the liver tissues were taken for immunohistochemical staining to calculate the proportion of Ki-67 positive cells, Western blot was used to detect the expression levels of X-box binding protein 1 (XBP1) and inositol-requiring enzyme 1α (IRE1α) proteins.Results① FLR/BW: On the 4th day and the 7th day after operation, the FLR/BW of the Sham group, PVL group, and ALPPS group increased in sequence at the same time, and the difference between the three groups was statistically significant (P<0.05). ② Ki-67 positive cell ratio: On the 2nd day after operation, the ratio of Ki-67 positive cells in the Sham group, PVL group, and ALPPS group increased sequentially, and the difference between the two groups was statistically significant (P<0.05). On the 4th day after operation, the ratio of Ki-67 positive cells in the PVL group and the ALPPS group were still higher than that of the Sham group (P<0.05). ③ Expression levels of XBP1 and IRE1α: On the 2nd and 4th postoperative day, the expression levels of XBP1 and IRE1α in the ALPPS group were higher than those in the Sham group and the PVL group (P<0.05). On the 7th day after surgery, the expression levels of XBP1 and IRE1α in the ALPPS group were higher than those in the Sham group (P<0.05), while compared with the PVL group, the expression level of XBP1 in the ALPPS group was still higher (P<0.05).ConclusionsALPPS-induced liver regeneration is more advantageous than traditional PVL in mice. It may be attributed to the obvious endoplasmic reticulum stress activation after ALPPS leading to the up-regulation of IRE1α-XBP1 expression, which is involved in the regulation of hepatocyte cell cycle and promotes hepatocyte proliferation, thus promoting rapid liver regeneration.
ObjectiveTo investigate the role of Peptidase domain containing associated with muscle regeneration 1 (PAMR1) in the proliferation, migration, and prognosis of hepatocellular carcinoma (HCC) through cellular experiments and clinical sample validation. Methods① Bioinformatics analysis was performed on datasets from the GEO public database to identify and screen for key genes, ultimately selecting PAMR1 for further study. Findings were validated using data from the TCGA database and six primary HCC surgical specimens prospectively obtained from the Army Characteristic Medical Center of Army Medical University from February 2024 to June 2024. ② PAMR1-overexpressing cell lines were established using HCC cell lines Huh7 and Lm3. Cells were transfected with either the recombinant plasmid pcDNA3.1(+)-PAMR1 as overexpression group or the empty vector pcDNA3.1(+) as negative control group. The effects of PAMR1 on HCC cell proliferation and migration were assessed using the Cell Counting Kit-8 (CCK-8) assay and wound healing assay, respectively. ③ Pathological specimens and clinical data were retrospectively collected from 61 patients with primary HCC who underwent surgical resection at the Army Characteristic Medical Center of Army Medical University between May 2019 and April 2020. The impact of PAMR1 expression on disease-free survival was evaluated. Results① PAMR1 was identified as a candidate gene through GEO database screening and was found to be downregulated in HCC tissues based on both TCGA data and the six HCC surgical specimens. ② The CCK-8 assay revealed that cell proliferation was significantly inhibited in the PAMR1 overexpression group compared to the negative control group (P<0.05). Similarly, the wound healing assay demonstrated reduced migratory capability in PAMR1 overexpression group (P<0.05). ③ Multivariate cox proportional hazards regression analysis of patient data indicated that high PAMR1 expression serves as an independent protective factor for disease-free survival in HCC (HR= 0.335, P=0.026). ConclusionPAMR1 serves as a crucial gene in HCC, high PAMR1 expression can significantly suppressing tumor cell proliferation and migration and indicates a favorable prognosis.
Objective To investigate the effect of ursolic acid on the proliferation and apoptosis of human osteosarcoma cell line U2-OS and analyze its mechanism. Methods Human osteosarcoma cell line U2-OS was divided into 4 groups, which was cultured with ursolic acid of 0, 10, 20, and 40 μmol/L, respectively. At 0, 24, 48, and 72 hours after being cultured, the cell proliferation ability was detected by cell counting kit 8 (CCK-8). At 48 hours, the effects of ursolic acid on cell cycle and apoptosis of U2-OS cells were measured by flow cytometry. Besides, the expressions of cyclin D1 and Caspase-3 were detected by real-time fluorescent quantitative PCR and Western blot. Results CCK-8 tests showed that the absorbance (A) value of each group was not significant at 0 and 24 hours (P>0.05); but the differences between groups were significant at 48 and 72 hours (P<0.05). Flow cytometry results showed that, with the ursolic acid concentration increasing, the G1 phase of U2-OS cells increased, the S phase and G2/M phase decreased, and cell apoptosis rate increased gradually. There were significant differences between groups (P<0.05). Compared with the 0 μmol/L group, the relative expressions of cyclin D1 mRNA and protein in 10, 20, and 40 μmol/L groups significantly decreased (P<0.05); whereas, there was no significant difference in relative expression of Caspase-3 mRNA between groups (P>0.05). However, with the ursolic acid concentration increasing, the relative expressions of pro-Caspase-3 protein decreased and the relative expressions of activated Caspase-3 increased; there were significant differences between groups (P<0.05). Conclusion Ursolic acid can effectively inhibit the proliferation of osteosarcoma cell line U2-OS, induce the down-regulation of cyclin D1 expression leading to G0/G1 phase arrest, increase the activation of Caspase-3 and promote cell apoptosis.
Objective To study the effects of adenosine 2A receptor activation on activation, proliferation, and toxicity of T lymphocytes stimulated by phytohemagglutinin (PHA) in vitro. Methods A model of activated T cells was established by stimulating the cells with PHA. Those T cells were treated with different concentrations of adenosine 2A receptors agonist (0.01 μmol/L, 0.1 μmol/L, 1 μmol/L, and 10 μmol/L CGS21680). The expressions of CD69, CD25 and proliferation of T cells were measured by fluorescent antibody stain and flow cytometry. ELISA method was used to detect IL-2 and INF-γ levels. Results All concentrations of CGS21680 significantly inhibited the expressions of CD25 and CD69 on PHA-stimulated T cells surface and proliferation of T cells （Plt;0.05， Plt;0.01）. IL-2 and INF-γ secreted by T cells were significantly suppressed, too （Plt;0.01）. Conclusion Activation of adenosine 2A receptor can effectively inhibit the activation, proliferation, and toxicity of T cells in vitro.
Objective To observe the effects of keratinocytes on proliferation and collagen secretion of fibroblasts. Methods The conditioned medium,collected from cultured keratinocytes, was added to the cultured fibroblasts as the tested groups(12.5％, 25% and 50％ groups) and DMEM as control group. The MTT, hydroxyproline coloricmetric method and flow cytometer were employed to measure the fibroblast proliferation, the collagen secretion andthe change of the cell cycle.Results In fibroblast proliferation, the absorbency(A) value of tested groups was significantly different from that of the control group (P＜0.01). A value increased as increasing concentration, there was statistically significant difference betweetheconcentrations of 25%,50% and the concentration of 12.5%(P＜0.01), but no statistically significant difference between the concentrations of 25% and 50%(P＞0.01). In collagen secretion, there was no statistically significant difference between the tested groups and the control group(P＞0.01), and between the tested groups(P＞0.01). In cell cycle, 50% of conditioned medium could make the fibroblast pass the limit of G1/S and S/G2 period, the cell rates of S,G2-M period increased. Conclusion The conditioned medium from keratinocytes can increase fibroblasts proliferation, have little effect on general collagen secretion.
Objective To investigate the feasibility of a dual-crosslinked injectable hydrogel derived from acellular musclar matrix (AMM) for promoting myoblasts proliferation and myogenic differentiation. Methods Firstly, hyaluronic acid was oxidized with NaIO4 and methylated to prepare methacrylamidated oxidized hyaluronic acid (MOHA). Then, AMM obtained by washing enzymatically treated muscle tissue was aminolyzed to prepare aminated AMM (AAMM). MOHA hydrogel and AAMM were crosslinked using Schiff based reaction and UV radiation to prepare a dual-crosslinked MOHA/AAMM injectable hydrogel. Fourier transform infrared spectroscopy (FTIR) was used to characterize MOHA, AAMM, and MOHA/AAMM hydrogels. The injectability of MOHA/AAMM hydrogel were evaluated by manual injection, and the gelation performance was assessed by UV crosslinking. The rheological properties and Young’s modulus of the hydrogel were examined through mechanical tests. The degradation rate of the hydrogel was assessed by immersing it in PBS. The active components of the hydrogel were verified using immunofluorescence staining and ELISA assay kits. The promotion of cell proliferation by the hydrogel was tested using live/dead staining and cell counting kit 8 (CCK-8) assays after co-culturing with C2C12 myoblasts for 9 days. The effect of the hydrogel on myogenic differentiation was evaluated by immunofluorescence staining and real time quantitative polymerase chain reaction (RT-qPCR). ResultsFTIR spectra confirmed the successful preparation of MOHA/AAMM hydrogel. The hydrogel exhibited good injectability and gelation ability. Compared to MOHA hydrogel, MOHA/AAMM hydrogel exhibited higher viscosity and Young’s modulus, a reduced degradation rate, and contained a higher amount of collagen (including collagen type Ⅰ and collagen type Ⅲ) as well as bioactive factors (including epidermal growth factor, fibroblast growth factor 2, vascular endothelial growth factor, and insulin-like growth factor 1). The live/dead cell staining and CCK-8 assay indicated that with prolonged incubation time, there was a significant increase in viable cells and a decrease in dead cells in the C2C12 myoblasts within the MOHA/AAMM hydrogel. Compared with MOHA hydrogel, the difference was significant at each time point (P<0.05). Immunofluorescence staining and RT-qPCR analysis demonstrated that the deposition of IGF-1 and expression levels of myogenic-related genes (including Myogenin, Troponin T, and myosin heavy chain) in the MOHA/AAMM group were significantly higher than those in the MOHA group (P<0.05). ConclusionThe MOHA/AAMM hydrogel prepared based on AMM can promote myoblasts proliferation and myogenic differentiation, providing a novel dual-crosslinked injectable hydrogel for muscle tissue engineering.