This article reported a case of primary coenzyme Q10 deficiency caused by a variation in the COQ4 gene. On the first day after birth, the neonate exhibited unexplained feeding difficulties, intermittent cyanosis, and respiratory and circulatory failure. Similar symptoms were observed in his sister, who passed away on the 9th day after birth but no pathogenic variant was detected in whole exome sequencing. After a pathogenic homozygous variant of COQ4 gene c.370G>A was detected in this patient using whole exome sequencing, his sister's result of whole exome sequencing was got and the same variant was found (identified as uncertain significance at that time), and both parents carried a heterozygous variant of c.370G>A. Supplement with clinical manifestations, the infant was diagnosed with coenzyme Q10 deficiency. The infant received respiratory and circulatory support, and after oral supplement of coenzyme Q10, the symptoms were improved. Subsequent follow-up examinations showed that the child had developed epilepsy and psychomotor retardation at about the age of one.

Objective To investigate the expression and mechanism of miR-1470 in plasma of diabetic retinopathy (DR) patients.Methods Thirty patients with DR (DR group),30 patients with diabetes (DM group) and 30 normal healthy subjects (normal group) were enrolled in the study.Three groups of subjects were taken 5 ml of venous blood,and total plasma RNA was extracted and purified.The differentially expressed miRNAs in the plasma of DR patients were screened by gene chip,and the results of gene chip detection were verified by reverse transcription polymerase chain reaction (RT-PCR).Bioinformatics was used to predict potential target genes for miRNA regulation,and miR-1470 and its target gene epidermal growth factor receptor (EGFR) were screened.Human retinal microvascular endothelial cells (hREC) were divided into normal group (sugar concentration 5.5 mmol/L) and high glucose group (sugar concentration 25.0 mmol/L).hREC was transfected into miR-1470 mimics to establish a miR-1470 high expression cell model,which was divided into blank control group,high expression group and negative control group.The expression of miR-1470 was detected by RT-PCR.The expression of EGFR protein was detected by Western blot.The measurement data of the two groups were compared using the independent sample t test.The comparison of the measurement data between the two groups was analyzed by ANOVA.The comparison between the measurement data of the groups was compared by multiple comparisons.Results The results of RT-PCR were consistent with those of the gene chip.The expression of miR-1470 in the plasma of the DR group,the DM group and the normal group was statistically significant (F=63.486,P=0.049).Compared with the DM group and the normal group,the expression of miR-1470 in the DR group was significantly decreased,and the difference was statistically significant (q=l 11.2,73.9;P＜0.05).The expression of miR-1470 in hREC in the high glucose group was significantly lower than that in the normal group (t=42.082,P=0.015).The expression of EGFR protein in hREC of high glucose group was significantly higher than that of normal group (t=-39.939,P=0.016).The expression of miR-1470 (F=637.069,P=0.000) and EGFR (F=122.908,P=0.000) protein expression in hREC of blank control group,negative control group and high expression group were statistically significant.Compared with the blank control group and the negative control group,the expression of miR-1470 in hREC of high expression group was significantly increased (q=329.7,328.8;P＜0.05),and the expression of EGFR protein was significantly decreased (q=242.5,234.6;P＜0.05).There was no significant difference in the expression of miR-1470 and EGFR protein in hREC between the negative control group and the blank control group (q=1.5,7.9;P＞0.05).Conclusion The expression ofmiR-1470 in the plasma of patients with DR is significantly down-regulated,and the increase of EGFR expression may be related to it.

Objective To explore repressive effects of transthyretitin (TTR) on the growth of human retinal endothelial cells (hREC) under high glucose and hypoxia environment. Methods hRECs were divided into 8 groups, including normal glucose group (5.5 mmol/L glucose), hypoxia group, high glucose group (25.0 mmol/L glucose), high glucose and hypoxia group, normal glucose group+TTR, normal glucose and hypoxia group+TTR, high glucose group+TTR, high glucose and hypoxia group+TTR. Flow cytometry was used to analyze cellular apoptosis. The expression level of Akt, p-Akt, eNOS, Bcl-2 and Bax protein were measured by Western blot. Results Hypoxia could induce apoptosis as the apoptosis rate of normal and hypoxia group was higher than normal group (χ2=25.360, P<0.05), high glucose and hypoxia group was higher that high glucose group (χ2=17.400, P<0.05). The cell apoptosis rate of high glucose and hypoxia group+TTR were increased significantly as compared with high glucose and hypoxia group (χ2=9.900, P<0.05). There was no statistically significant difference on the cell apoptosis rate between normal group and high glucose group, normal group+TTR and normal group, high glucose group+TTR and high glucose group, normal and hypoxia group+TTR and normal and hypoxia group (P>0.05). Western blot showed that the expression of Akt did not change significantly in all eight groups(F=2.450, P>0.05). Compared to normal group, the expression of p-Akt, eNOS, Bcl-2 in normal and hypoxia group were decreased (t=9.406, 5.306, 4.819), and the expression of Bax (t=-4.503) was increased (P<0.05). Compared to high glucose group, same trend was found in high glucose and hypoxia group (t=8.877, 7.723, 6.500, -14.646; P<0.05). The expression of p-Akt in normal and hypoxia group+TTR was higher than normal and hypoxia group (t=-5.024, P<0.05) ,but there was no difference on the expression of eNOS, Bcl-2, Bax between these two groups (t=-2.235, -2.656, -0.272;P>0.05). Compared to high glucose and hypoxia group, the expression of p-Akt and Bcl-2 in high glucose and hypoxia group+TTR were decreased (t=4.355, 4.308; P<0.05), the expression of Bax was increased (t=-4.311, P<0.05), and there was no difference on the expression of eNOS between these two groups (t=-1.590, P>0.05). There was no statistically significant difference in the expression of p-Akt, eNOS, Bcl-2, Bax between high glucose group and normal group (t=-3.407, -4.228, -4.302, -2.076; P>0.05), normal group+TTR and normal group (t=-4.245, -4.298, -2.816, -1.326; P>0.05), high glucose group+TTR and high glucose group (t=4.016, -0.784, 0.707, -0.328; P>0.05). Conclusion Under high glucose and hypoxia, transthyretitin suppress the growth of hREC through Akt/Bcl-2/Bax, but not Akt/eNOS signaling pathway.

Objective To investigate the expression of HSPA9 in hepatocellular carcinoma(HCC) and its relationship with clinicopathological features and prognosis.Methods Forty-nine cases HCC treated by operative resection and follow up data in our hospital from January 2006 to January 2010 were retrospectively analyzed.Immunohistochemistry was performed to determine the expression of HSPA9 in HCC and paratumor tissues.The relationship between HSPA9 expression and clinicopathological features and prognosis was statistically analyzed.Results The HSPA9 protein expression in tumor tissue was higher that that in the paratumor tissue(t=6.601,P＜0.01),moreover the over expression of HSPA9 was significantly correlated with lymph node metastasis (P =0.005),TNM-stage(P =0.015),tumor differentiation (P =0.033),microvascular invasion (P =0.009) and recurrence (P =0.047).In the survival analysis results,the patients with over expression of HSPA9 had a much lower total survival rate(P=0.002)and much higher postoperative cumulative recurrence rate(P =0.003).There were significant differences in TNM-stage,microvascular invasion,lymph node metastasis,tumor differentiation and HSPA9 staining for overall survival and cumulative recurrence rate based on a univariate analysis(P＜0.05).Conclusion HSPA9 has over expression in HCC.The over expression of HS-PA9 is closely related to invasion and metastasis pathological features and can serve as an independent prognostic risk factor for predicting the prognosis of HCC.

Objective To explore the effect and mechanism of fragile site WWOX gene on regulating proliferation of gallbladder cancer cells in vitro. Methods The pcDNA3.0 - WWOX recombinant plasmid which was previous successfully built was transfected to GBC-SD cells and empty carrier by liposome medium. Liposome and GBC-SD were served as the negative control and the blank control,respectively. After 48 hours transfection, inverted microscope was used to observe the changes of gallbladder cancer cells' morphology,MTT and BrdU were used to detect the proliferation level of gallbladder cancer cells,and flow cytometry instrument was used to detect the change of the cell proliferation cycle. Results The results of inverted microscope shown: the number of GBC-SD cells in pcDNA3.0-WWOX group decreased significantly,the suspension cells and cell debris increased,while cells in the vector control,NC and Mock groups were in normal proliferation state. MTT test showed the proliferation levels of GBC-SD cells in pcDNA3.0-WWOX group was lower than those in the control group in 24 h,48 h,72 h,96 h and 120 h,and the differences were statistically significant(P < 0.05). The cell proliferation activity in the pcDNA3.0-WWOX group was obviously inhibited over time. BrdU detection results showed the cell proliferation rate of pcDNA3.0 - WWOX group was(0.44±0.03),while that in the three control groups was(0.78±0.02), (0.81±0.01)and(0.85±0.01),respectively. It showed that cell proliferation activity in pcDNA3.0-WWOX group was lower than the control groups,and the difference was statistically significant(P < 0.05). Cell cycle detection showed the cells increased in G0/G1 phase and decreased in G2/M and S phases of pcDNA3.0-WWOX group. The cell apoptosis rate was significantly higher and the proliferation index was significantly lower than those of the control groups(P < 0.05). However,there were no significant differences among the three control groups(P > 0.05). Conclusion The overexpression of WWOX gene in vitro could effectively inhibit the proliferation activity of gallbladder cancer cells. WWOX might participate in the development of the malignant biological behavior of gallbladder cancer cells. It is expected to become a new potential target for the gene therapy to gallbladder cancer.

Objective To investigate paclitaxel and ezrin-siRNA therapy for metastatic liver cancer.Methods Human metastatic liver cancer cells MHCC97-H were divided into four groups in vitro and in vivo,including control cells,exposure to paclitaxel alone,ezrin-siRNA alone,and the combination of paclitaxel and ezrin-siRNA.Nude mice lung lesions were formed by tail vein injection of tumor cells.The tumor cells'apoptosis rate,wound healing ability,the ability to cross an artificial membrane,and the numbers of metastatic lung lesions were measured.Results The apoptosis rate in normally cultured MHCC97-H tumor cells was 2.52％,compared to the other three groups at 11.66％,2.35％,7.38％ respectively.The wound healing ability was best in the negative control and weakest in cells treated with combination paclitaxel and ezrin-siRNA.The numbers of cells which could cross the artificial membrane in the negative,paclitaxel,ezrin-siRNA,and combination paclitaxel and ezrin-siRNA group were 52.5 ± 3.9,32.2 ± 3.2,26.6 ± 2.4,and 19.4 ± 1.1 respectively.The numbers of metastatic lung lesions in the nude mice (n =40) were 12.5 ±2.4,8.2 ± 1.5,7.0 ± 1.3,and 3.8 ± 0.3 respectively.Conclusions The ezrin-siRNA combined with paclitaxel therapy demonstrated a greater efficacy over single agent therapy for depressing liver cancer growth,motility,aggression,and metastasis in vitro and in vivo.

Objective To establish the method of combined hepatocyte growth factor (HGF) with epidermal growth factor (EGF) cultured human gallbladder epithelial cells(HGBECs) in vitro .Methods The epithelial layer was peeled away from human gallbladder ,epithelial layer were digested with collagenase Ⅳ and scraped repeatedly .HGBECs were isolated and seeded in cell cul-ture plates containing medium supplemented with or without 10 ng/mL EGF or with 10 ng/mL HGF and 10 ng/mL EGF respec-tively .Then the morphologic changes of the cells were observed and taken photos with inverted phase contrast microscope ,and counted number of cells ,MTT assay detected vigor of cells in different groups .Results The number of the HGBECs of the HGF+EGF group was obviously more than the EGF group ,the duration of the HGBECs of the HGF+ EGF group was obviously longer than the EGF group(19 .3 ± 2 .5)d vs .(14 .2 ± 2 .4)d ,P< 0 .05 .And the HGBECs of the group with HGF+ EGF had better cell vigor .Conclusion HGF combines with EGF added to medium can obviously promote the proliferation of HGBECs and prolong the duration and stabilize morphology of HGBECs in vitro .

Aged ; Airway Obstruction ; chemically induced ; Analgesics, Opioid ; adverse effects ; Anesthesia, General ; adverse effects ; Fentanyl ; administration & dosage ; Humans ; Male ; Muscle Rigidity ; chemically induced

Objective Via targeted inhibition of oncogene Bmi-1 expression by RNAi interfering technology in vitro, to observe its effect on the proliferation and cell cycle of gallbladder cancer cells. Methods Four miRNABmi-1 recombinant plasmids were constructed according to different Bmi-1 sites. RT-PCR and Western blot were used to mRNA and protein expression of Bmi-1 in gallbladder cancer cells were measured by RT-PCR and Western blot. mRNA and protein expression of Bmi-1 in gallbladder cancer cells. The most effective interfering plasmids in the miRNABmi-1 groups were transfected into GBC-SD cells. Cell proliferation and cell cycle were analyzed 48 h after transfection by BrdU and flow cytometry. Results Bmi-1mRNA expression in miRNAbmi1-1,-3 and-4 was significantly lower than the control group (P<0.05);and Bmi-1 protein expression in miRNAbmi1-2,-3 and-4 was significantly lower than the control group (P<0.05). The recombinant plasmid in miRNAbmi1-4, with the strongest inhibitive effect of Bmi-1mRNA and protein expression, was transfected into GBC-SD cells,then the cell proliferation rate (46.63 ± 5.31) was significantly lower in mRNABmi1-4 group than the control groups (P<0.05);G0/G1 phase cells increased (72.20 ± 1.71) and G2/M and S phase cells decreased (18.30 ± 7.21, 9.50 ± 6.01) in miRNABmi1-4 group. Both were significantly different from the control groups (P<0.05). Conclusions Targeting and silencing Bmi-1 expression can effectively inhibit the proliferation of GBC-SD cells and restrain the cell cycle atin G0/G1 phase. Bmi-1 gene may be a novel target for geneic therapy of gallbladder carcinoma.

BACKGROUND:In vitro and in vivo studies of cel response to a variety of mechanical loadings have demonstrated the stimulation of bone formation by loads. However, the effects of different mechanical strains on the same cel s have never been adequately studied by far.
OBJECTIVE:To investigate the effects of different mechanical strains on rat bone marrow mesenchymal stem cel s.
METHODS:Rat bone marrow mesenchymal stem cel s were isolated and cultured in vitro. Bone marrow mesenchymal stem cel s were subjected to different stimulations including dynamic stretch, static stretch and hybrid stretch through the use of custom-made mechanical stretch device. Cel ular proliferation, alkaline phosphatase activity and mRNA expression of Runx2 of bone marrow mesenchymal stem cel s were detected and the secretion of osteocalcin was evaluated under three different stretch modes respectively.
RESULTS AND CONCLUSION:Compared to the control group, cel proliferation increased by 18.67%, however, alkaline phosphatase activity, Runx2 expression and osteocalcin secretion were not changed obviously in the static stretchgroup. Compared to the control group, alkaline phosphatase activity, Runx2 expression and osteocalcin secretion increased by 60.33%, 49.67%and 48%respectively;however, cel proliferation was inhibited, in the dynamic stretch group. Compared to the control group, cel proliferation was slightly, but not significantly, increased in the hybrid stretch group, and the alkaline phosphatase activity, Runx2 expression and osteocalcin secretion increased although the increases were not as apparent as those in the dynamic stretch group. These findings suggest that static mechanical strain can significantly promote cel proliferation, the dynamic mechanical strain more greatly promotes osteogenic differentiation of bone marrow mesenchymal stem cel s, and the hybrid mechanical strain promotes the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cel s.