ObjectiveTo investigate whether Osteoglycin (OGN) gene inhibits the proliferation of Luminal breast cancer cells by up-regulating the expression of estrogen receptor (ER). Methods① Ualcan online database was used to analyze the expression of OGN in the breast cancer, and Kaplan-Meier Plotter was used to analyze the effect of OGN on the prognosis of patients with breast cancer. The median OGN mRNA expression level was taken as the cut-off point for high or low OGN expression. ② The expression of OGN mRNA in the Luminal breast cancer tissue of clinical case was examined using real time quantitative PCR (qRT-PCR). ③ Up-regulation of OGN expression in the Luminal breast cancer cell lines MCF-7 and T47D cells by transfection of overexpressing OGN plasmid, the expressions of OGN and ER were detected by qRT-PCR and Western blot, respectively. CCK8 assay and colony formation assay were applied to detect the cell proliferation and colony formation of Luminal breast cancer cells. ④ siRNA transfection was used to interfere with the expression of ER (ESR1) of breast cancer cells in the overexpressing OGN of breast cancer cells, then the CCK8 assay was used to detect the proliferation ability of the breast cancer cell lines after down-regulating the expression of ER in the overexpressing OGN patients. Results① The results of Ualcan online database showed that the expression of OGN mRNA in the breast cancer tissues of different types of breast cancer was lower than that in the normal breast tissues (P<0.001), and which was highest in the Luminal breast cancer tissues (P<0.001). The Kaplan-Meier Plotter prognosis analysis showed that in all breast cancer or Luminal breast cancer patients, the prognosis of patients with high OGN expression was better than those with low OGN expression (P=0.000 14, P=0.001 80). ② The OGN mRNA expression was decreased in the 30 Luminal breast cancer samples as compared with the corresponding adjacent normal breast tissues (t=4.774, P=0.000 019). ③ The expressions of OGN and ER in the MCF-7 and T47D cells were up-regulated after transfection of overexpressing OGN plasmid (P=0.000 002, P=0.000 001). The cell proliferation was inhibited (P<0.05) and the number of cell clones was decreased significantly (P<0.05). ④ After transient transfection of siRNA interfered with breast cancer cell lines of overexpressing OGN, ER mRNA level decreased (P<0.05), and cell proliferation ability increased significantly (P<0.05). Conclusion OGN could exert a tumor suppressor effect in Luminal breast cancer by mediating expression of ER.
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.
Objective To investigate the relationship of the expression between heat shock protein (HSP) 70 and 90, and Survivin and its effects on the proliferative activity in retinoblastoma (RB) cells. Methods Expression of Survivin, HSP70 and 90, and Ki-67 in conventional paraffin samples from 43 patients with RB and 6 healthy people was detected by streptavidin-biotin peroxidase (SP) immunohistochemical method. Ki67 labeling index was used to evaluate the proliferative activity in RB. Results In 43 cases of RB, positive expression of HSP70 and 90 and Survivin was found in 28 (65.12%), 37 (86.05%) and 27 (62.79%) cases, respectively. None of the 6 normal retinal tissue expressed HSP70, HSP90 or Survivin. Positive expression of Survivin was more frequent in positive expressions of HSP90 than that in negative expressions of HSP90 (P＜0.05). Ki67 labeling index was higher in positive expressions of HSP90 and positive expressions of Survivin than that in their negative expressions respectively (P＜0.05). Meanwhile, higher Ki67 labeling index was found in positive HSP90Survivin expressions than that in negative HSP90Survivin expressions and those cases where only HSP90 or Survivin was found (P＜0.05). Expression of HSP70 did not correlate with that of Survivin, nor had any significant effect on Ki67 labeling index (P＞0.05). Expression of HSPs and Survivin and Ki67 labeling index did not correlate with histological types (P＞0.05). Conclusion Expression of HSP90 correlates with that of Survivin in RB. Co-existence of Survivin and HSP90 probably plays an important role in the genesis of RB.
Objective To investigate the role of chemokine receptor CXCR7 in the development and progression of pancreatic carcinoma. Methods The short hairpin RNA (shRNA) targeting CXCR7 was designed and delivered into AsPC-1 pancreatic carcinoma cells to knock down CXCR7 expression. The cell proliferation, cell cycle, and apoptosis after CXCR7 knockdown was determined by MTT and flow cytometry, respectively. The invasive ability of pancreatic carcinoma cells was evaluated by using the Transwell system. Results Compared with the blank control group (BC group), transfection of AsPC-1 cells with CXCR7-shRNA resulted in a significantly decreased expression of CXCR7 at both mRNA and protein levels (P<0.05), and the ability of proliferation and invasion significantly decreased (P<0.05). Knockdown of CXCR7 also significantly increase apoptosis (P<0.05), induce cell cycle arrest at G0/G1 phase (P<0.05). Conclusions Taken together, the present study showes that the knockdown of CXCR7 expression may play an important role in pancreatic carcinoma development, invasion, and metastasis, CXCR7 may be a potential therapeutic target for the treatment of pancreatic carcinoma.
Tostudytheexpressionofproliferatingcellnuclearantigen(PCNA)inhumangastricneoplasmanditsclinicalsignificance.TheexpressionofPCNAwassemiquantitativelyanalysedimmunohistochemically(ABC)in10gastricadenomatouspolypand94gastriccarcinomas.Results:①PCNAlabelingindex(LI)showedasignificantdifferentiationindifferentpathologicstate(Plt;0.01),②PCNALIingastriccarcinomawasindepednetofsexandage(Pgt;0.05),butcorrelatedwiththegrowingmannerofthetumor,tumordifferentiation,serosalinfiltration,nodalmetastasisandclinicalstages(Plt;0.05),③ThecorrelationwasfoundbetweenPCNALIandprognosisofgastriccarcinoma(Plt;0.05).AmoderategradeofPCNAexpressiongenerallyhadabetterprognosis.Conclusions:TheseresultssuggestthatPCNALIisafairlygoodindextoindicatebiologicbehaviorandprognosisingastriccarcinoma.
Objective To analyze MC3T3E1 cell morphology, prol iferation, and osteogenic differentiation in fibrin gel (FG) so as to lay a fundament for use of FG in tissue engneering. Methods MC3T3E1 cells were incubated in three concentrations (20, 10 and 5 mg/mL)of FG as the experimental groups (groups A, B and C) and in the common medium culture as the control group (group D). The cell morphology and distribution in FG were observed by inverted phase contrast microscope and confocal laser scanning microscope at different time. The cell prol iferation was assessed by fluorospectrophotometer. The alkal ine phosphatase (ALP) activity was detected by automatic biochemistry analyses and von Kossa staining was used to analyze calcium salts mineralization. RT-PCR was used to analyze the ALP and bone sialoprotein (BSP)mRNA expression at 14 and 21 days. Results In groups A, B and C, the MC3T3E1 cells had long processes which connected each other and formed network; but fusiform or cube cells were observed in group D at 21 days. The fluorescence intensity was increased gradually with time, was the highest at 14 days and the lowest at 28 days in group D; it was highest in groups A, B and C at 28 days, there were statistically significant differences when compared with group D (P lt; 0.05). The ALP activity was increased gradually with time, and it was the highest at 28 days in group D and at 21 days in groups A and B, there were significant differences (P lt; 0.05), no statistically significant differences compared with group D at other time points (P gt; 0.05). The mineral ization nodus were seen at 21 and 28 days in group A, but no mineral ization nodus was seen in group D at 28 days. The RT-PCR results showed the mRNA expressions of ALP and BSP were enhanced in group A when compared with group D (P lt; 0.05). Conclusion The osteogenic differentiation was most obvious and cell prol iferation was most active after 21 days of incubation in FG.
ObjectiveTo establish the system of isolation, cultivation, and identification of the neural stem cells (NSCs) from subventricular zone (SVZ) of neonatal mice so as to seek for the appropriate seed cells for potential therapeutic interventions of neurological disorders. MethodsNSCs were isolated enzymatically and mechanically from SVZ of neonatal mice and cultured. The cellular morphology was observed by inverted microscopy. Immunocytochemical stainings of anti-Nestin and anti-SOX-2 were used to identify NSCs of passage 3. To study the differentiation of NSCs, NSCs were plated into 24-wells in the medium supplemented without epidermal growth factor (EGF) and basic fibroblastic growth factor (bFGF) for 3 or 7 days. To compare the differentiation and proliferation potential of NSCs with different cultivation time, the BrdU pulse-labeling method and MTT test were used. To identify neurons and astrocytes, the anti-β-tubulin Ⅲ (Tuj-1) and anti-glial fibrillary acidic protein (GFAP) staining were used. ResultsThe cells of the SVZ can be isolated and cultured in vitro, and these cells began to form neurospheres after cultured for 3 days at primary passage. While cultured for 7 days, these cells formed more neurospheres, and the volume of the neurospheres became bigger than neurospheres cultured for 3 days. In addition, after cultured for 7 days, the phenomena of fusion of neurospheres and adherent differentiation of neurospheres were observed under inverted microscope. These cells were provided with the typical phenotype of NSCs. The immunofluorescence staining results revealed that these cells showed positive immunoreactivity to Nestin and SOX-2. During the 4 hours BrdU pulse, the number of proliferated NSCs cultured for 3 days (75.817±2.961) was significantly higher than that of NSCs cultured for 7 days (56.600±4.881) (t=3.366, P=0.028). The results of MTT assay revealed that the absorbance (A) value of NSCs cultured for 3 days (0.478±0.025) was significantly higher than that of NSCs which were cultured for 7 days (0.366±0.032)(t=2.752, P=0.011). After cultivated without EGF and bFGF, the percentage of Tuj-1 and GFAP positive cells in NSCs was 23.1%±3.7% and 23.7%±3.8% for 3 days and was 40.1%±3.6% and 37.1%±4.5% for 7 days, respectively, all showing significant differences (t=3.285, P=0.030; t=3.930, P=0.017). ConclusionThe NSCs from SVZ of neonatal mice have potentials of self-renewal and multipotential differentiation in vitro. With different cultivation time, the potentials of proliferation and differentiation of NSCs are different.
Objective To observe the effects of SARS-CoV-2 infection on the morphology, proliferation, apoptosis, cell cycle, and immune response function of mouse retinal photoreceptor cells (661w cells). MethodsA cell experiment. Logarithmic growth phase 661w cells were cultured in vitro and transfected with angiotensin-converting enzyme 2 (ACE2) overexpressing lentivirus to construct ACE2 overexpressing 661w cells that could be infected with SARS-CoV-2 pseudovirus (hereafter referred to as ‘pseudovirus’). The 661w cells were divided into three groups: the normal group (untreated), the siACE2 group (overexpressing ACE2 and not infected with the pseudovirus) and the infected group (overexpressing ACE2 and infected with the pseudovirus), in which the infected group was 5 TU/ml pseudovirus group, 15 TU/ml pseudovirus group, 30 TU/ml pseudovirus group and 50 TU/ml pseudovirus group, and the cells were infected with the pseudovirus for 12, 24, 48 and 72 h, respectively. The infected group was infected with 5 TU/ml pseudovirus group, 15 TU/ml pseudovirus group, 30 TU/ml pseudovirus group and 50 TU/ml pseudovirus group, respectively, for 12, 24, 48 and 72 h. Fluorescence microscopy was used to observe the transfection efficiency of ACE2; protein immunoblotting (Western blot) was used to detect the relative expression level of ACE2 in the cells; light microscope was used to observe the morphology of the cells in the normal and the infected groups; cell proliferation was detected by Cell Counting Kit-8 (CCK8) assay; flow cytometry was used to detect the cell cycle; Western blot and real-time quantitative polymerase chain reaction (qPCR) were used to detect the relative expression of interleukin-6 (IL-6), tumour necrosis factor-α (TNF-α), B lymphocytoma-2 (Bcl-2), Bcl-2-associated X-protein (Bax) proteins and mRNA in the cells of siACE2 group, infected group (30 TU/ml pseudovirus group); qPCR was used to detect the relative expression of nuclear factor (NF)- κB1 and NF-κB2, as well as NF- kB enhancer (P65) and precursor protein (P100) in cells of the siACE2 group and the infected group (30 TU/ml pseudovirus group). One-way ANOVA was used for comparison between multiple groups; t-test was used for comparison between two groups. Results Compared with the siACE2 group, the cells in the infected group showed different degrees of crumpling, and with the increase of the concentration and time of pseudovirus induction, the crumpling of the cells worsened, and the number of cells decreased. Compared with the normal group, the cells in the infected group showed a gradual decrease in cell viability with the prolongation of pseudovirus induction time, and the difference was no statistically significant (F=0.840, 0.412, 1.498, 1.138; P＞0.05), and the apoptotic index of the cells induced in the 30 and 50 TU/ml pseudovirus group was significantly elevated, and the difference was statistically significant (F=2.523, 6.716, 3.477, 3.421; P＜0.05). At 72 h of pseudovirus induction, compared with the siACE2 group, the G1 phase cells in the 30 TU/ml pseudovirus group were significantly increased, and the difference was statistically significant (t=3.812, P＜0.05); the relative expression of IL-6, TNF-α, Bax protein and mRNA in the cells was up-regulated (t=7.601, 6.039, 3.088, 5.193, 6.427, 7.667; P＜0.05), the relative expression of Bcl-2 protein and mRNA was down-regulated (t=3.614, 6.777; P＜0.05), and the relative expression of NF-κB1, NF-κB2, P65, and P100 mRNA was significantly up-regulated with statistically significant differences (t=3.550, 3.074, 3.307, 4.218; P＜0.05). ConclusionSARS-CoV-2 infection may inhibit photoreceptor cell proliferation, promote apoptosis and cycle blockade by activating the NF-κB signalling pathway.
Objective To evaluate the expression of reactive oxygen species (ROS), glutataione (GSH), total superoxide dismutase (T-SOD), total antioxidation capacity (T-AOC), thioredoxin reductase (TrxR) under the intervention of hedysarum polysaccharides-1 (HPS-1) in A549 cells. Methods After treated by HPS-1 in different doses (50 mg/L, 100 mg/L, 200 mg/L, respectively), the viability of cell lines was detected by MTT method under microscope. The apoptosis of cell lines was detected by flow cytometry (FCM). The expressions of ROS, GSH, T-SOD, T-AOC, and TrxR in cell supernatant were measured by chemiluminescence method. Results Determined by MTT/FCM/ELISA, the results showed that different doses of HPS-1 could inhibit the proliferation and promote the apoptosis of A549 cells (allP<0.05). The expression levels of GSH, T-SOD, T-AOC, and TrxR were significantly decreased (allP<0.05) and the expression levels of ROS and MDA were significantly increased (allP<0.05) in a concentration-dependent manner in A549 cells treated with HPS-1, and these effects were significantly weakened in A549 cells with time extending (allP<0.05). Conclusion HPS-1 has a markedly effect on inhibiting cellular proliferation and inducing cellular apoptosis of lung adenocarcinoma A549 cells, which may be associated with the change of oxidation/antioxidant.
OBJECTIVE: To observe the effects of hyaluronic acid (HA) and basic fibroblast growth factor (bFGF) on the proliferation of the cells from medial collateral ligament (MCL) and anterior cruciate ligament (ACL) cells. METHODS: The MCL cells and ACL cells of mature New Zealand white rabbit were cultured, while HA, bFGF or HA and bFGF were added to the cell culture media, the cellular proliferation was assayed by MTT method. RESULTS: HA only had no effect on the preoliferation of ACL cells, but had a small stimulatory effect on the proliferation of MCL cells. The addition of 1 ng/ml bFGF enhanced the proliferation of both MCL and ACL cells significantly, and this enhancement was maximal in the concentration of 50 ng/ml. However, the enhancement of proliferation of MCL and ACL cells could be achieved when the combination of HA in concentration of 100 micrograms/ml and bFGF in concentration of 100 ng/ml. CONCLUSION: It is evident that bFGF can enhance the proliferation of the ligament cells. HA can maintain the normal growth of ACL cells with no effect on the proliferation of the cells, while HA has a small stimulatory effect on the proliferation of MCL cells. However, when bFGF is coordinated with HA, more improvement of cellular proliferation can be achieved. HA can be used as a potential carrier for bFGF to enhance the healing of ligamentous tissue injuries.