Long-term treatment with an agonist of peroxisome proliferator-activated receptor (PPAR)-γ is associated with bone fractures in the clinical practice. However, the mechanisms underlying the fractures are not fully understood. This study was aimed to examine the effect of rosiglitazone (an agonist of PPAR-γ) of different doses on the proliferation, differentiation, and transforming growth factor beta 1 (TGF-β1)-induced expression of connective tissue growth factor (CTGF) in primary rat osteoblasts in vitro. Osteoblasts were isolated from newly born SD rats and treated with different doses of rosiglitazone (0-20 μmol/L). The proliferation and differentiation of osteoblasts were measured by MTT assay and NPP assay, respectively. The expression of CTGF was determined by RT-PCR and Western blotting. The results showed that most isolated osteoblasts displayed strong alkaline phosphatase (ALP) activity and treatment with different doses of rosiglitazone did not affect their proliferation, but significantly inhibited the differentiation of osteoblasts in a dose-dependent manner. Moreover, treatment with different doses of rosiglitazone significantly reduced the TGF-β1-induced CTGF mRNA transcription and protein expression in a dose-dependent manner in rat osteoblasts. It was concluded that the activation of PPAR-γ may inhibit the differentiation of osteoblasts by reducing the TGF-β1-induced CTGF expression in vitro.

Objective To investigate the inhibitory effects of RNA interference(RNAi)on the expression of matrix metalloproteinase-9(MMP-9)gene and invasiveness and adhesion of ovarian cancer cells.Methods Four groups of different specific target sequence in coding region of MMP-9 and one non- specific sequence were chosen,which were Sitel,Site2,Site3,Site4 and Site5.Small interference RNA (siRNA)expression cassettes(SEC)were constructed by PCR and transfected into ovarian cancer HO- 8910PM cells.RT-PCR and western blot were used to detect mRNA and protein expression of MMP-9 gene; the abilities of invasion and adhesion were detected by Matrigel invasion assay and cell adhesion assay. Results The expression of MMP-9 was inhibited and the inhibitory effects of different sequence were varied.The mRNA expression was 0.64?0.06,0.47?0.07,0.55?0.10 in Sitel,Site2,Site3 group, and protein expression was 0.30?0.09,0.27?0.08,0.37?0.12,respectively.Site2 group had the most efficient inhibitory effect,followed by Sitel and Site3 groups.Cell growth curve revealed that cell growth was significantly inhibited in Site2 group.Invasiveness and adhesion were significantly reduced,the inhibitory rate on invasion in Site1,Site2,Site3 groups were 50.0%,50.0% and 37.5%,respectively;the inhibitory rate on adhesion in Site1,Site2,Site3,Site4 groups were 43.8%,48.8%,33.9%,24.2% at 60 min and 41.6%,40.2%,35.1%,16.0% at 90 min,respectively.Conclusions RNAi exists in ovarian HO-8910PM cells.MMP-9 siRNA can specifically down-regulate MMP-9 expression and lead to the inhibition of invasiveness and adhesion in ovarian cancer cells.

Objective: To provide reference for clinical pharmacists to write case analysis work .Methods: The reviews of 185 case analysis work of clinical pharmacists from the training bases all over the country were collected .Combined with the practical expe-rience of clinical teaching , the reviews were analyzed and discussed .Results:The problems in title , introduction , drug use analysis , summary and experience of the case analysis work were pointed out , and some improvement suggestions were provided , especially those for the improvement of case analysis evaluation form .Conclusion:Although the case analysis work has been screened by the teachers of training bases , the problems are still outstanding .The rationalization proposals provided in the paper maybe help improve the quality of case analysis work .

Objective To summarize the diagnosis,treatment and prevention of dysuria within 6 months after the suprapubic transvesical prostatectomy. Methods Twenty-four cases were retrospectively reviewed,including the data on the diagnosis, treatment and prognosis. Results Twenty-one of the 24 cases had received surgery. There were 9 cases with bladder neck stricture,9 cases with posterior urethra stricture and 3 cases with of remnant glands. Nine cases received transurethral bladder neck incision, 9 received open surgery ( bladder neck,posterior urethral incision) and 3 received transurethral resection of the prostate (TURP) plus bladder neck incision. The other 3 diagnosed as detrusor weakness were conservatively treated by indwelling catheter and they were improved one months later. None of these patients was readmitted into hospital for dysuria within 6 months after treatment. Conclusion The recurrence of dysuria post prostatectomy mostly ( 75% ) occurred within 6 months after surgery. A majority of these patients need a second surgery. To avoid a second surgery for postoperative dysuria, much attention should be paid to the operating technique and postoperative management.

Long-term treatment with an agonist of peroxisome proliferator-activated receptor (PPAR)-γ is associated with bone fractures in the clinical practice. However, the mechanisms underlying the fractures are not fully understood. This study was aimed to examine the effect of rosiglitazone (an agonist of PPAR-γ) of different doses on the proliferation, differentiation, and transforming growth factor beta 1 (TGF-β1)-induced expression of connective tissue growth factor (CTGF) in primary rat osteoblasts in vitro. Osteoblasts were isolated from newly born SD rats and treated with different doses of rosiglitazone (0-20 μmol/L). The proliferation and differentiation of osteoblasts were measured by MTT assay and NPP assay, respectively. The expression of CTGF was determined by RT-PCR and Western blotting. The results showed that most isolated osteoblasts displayed strong alkaline phosphatase (ALP) activity and treatment with different doses of rosiglitazone did not affect their proliferation, but significantly inhibited the differentiation of osteoblasts in a dose-dependent manner. Moreover, treatment with different doses of rosiglitazone significantly reduced the TGF-β1-induced CTGF mRNA transcription and protein expression in a dose-dependent manner in rat osteoblasts. It was concluded that the activation of PPAR-γ may inhibit the differentiation of osteoblasts by reducing the TGF-β1-induced CTGF expression in vitro.
Animals ; Animals, Newborn ; Blotting, Western ; Cell Differentiation ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Connective Tissue Growth Factor ; genetics ; metabolism ; Dose-Response Relationship, Drug ; Gene Expression ; drug effects ; Hypoglycemic Agents ; pharmacology ; Osteoblasts ; cytology ; drug effects ; metabolism ; PPAR gamma ; agonists ; metabolism ; Rats, Sprague-Dawley ; Reverse Transcriptase Polymerase Chain Reaction ; Thiazolidinediones ; pharmacology ; Transforming Growth Factor beta1 ; pharmacology

OBJECTIVETo investigate the effects of tumor necrosis factor alpha (TNF-alpha) on the growth of rat osteoblasts. To find out the mechanisms that TNF-alpha regulates the growth of osteoblasts.

METHODSTo assay osteoblasts proliferation by MTT. To assay alkaline phosphatase (ALP) activity of osteoblasts by PP-nitrophenyl phosphate (PNPP).

RESULTSThe osteblasts proliferation and the ALP activity decreased in treatment groups, and the significantly lower levels were observed in above 50 ng/mL groups (P<0.05).

CONCLUSIONTNF-alpha restrained osteoblasts proliferation and differentiation, and the effects were more significant in above 50 ng/mL groups.

Alkaline Phosphatase ; Animals ; Cell Differentiation ; Cell Proliferation ; Osteoblasts ; Rats ; Tumor Necrosis Factor-alpha

OBJECTIVETo observe the inhibitory effects of antisense MMP-9 oligodeoxynucleotides on invasiveness and adhesion ability in vitro of ovarian cancer cells, and to investigate the mechanisms of action.

METHODSMMP-9 antisense oligonucleotides were transfected by lipofectinmin into ovarian cancer cell line HO-8910PM cells expressing MMP-9 induced with fibronectin. RT-PCR, Western blot and gelatin zymography were used to detected MMP-9 expression of mRNA and protein and enzymatic activity. The ability of invasion and migration of ovarian cancer cells was assayed in Transwell cell culture chamber. Cell adhersion assay was carried out in a microculture well pre-coated with fibronectin.

RESULTSMMP-9 expressions of mRNA and protein were significantly decreased in the antisense-transfected cells. Comparing with the control group, the inhibition rate was 34. 8% and 42. 5% , respectively (P <0. 05). Its gelatin enzymatic activity was inhibited. Matrigel invasion assay and Transwell migration assay revealed markedly reduction in invasion and migration for the antisense group. The inhibition rates were 22. 4% and 24. 8% , respectively. The adhesion ability was also reduced. The inhibition rates were 49. 8% and 38. 3% at 60 min and 90 min, respectively.

CONCLUSIONMMP-9 down-regulation can significantly inhibit the ability of invasion and attachment of ovarian cells in vitro. MMP-9 may play an important role in invasion and metastasis of ovarian cells and potentially be a molecular target of blocking invasion and metastasis of ovarian cancer.

Blotting, Western ; Cell Adhesion ; genetics ; physiology ; Cell Line, Tumor ; Cell Movement ; genetics ; physiology ; Down-Regulation ; Female ; Humans ; Matrix Metalloproteinase 9 ; genetics ; metabolism ; Neoplasm Invasiveness ; Oligodeoxyribonucleotides, Antisense ; genetics ; Ovarian Neoplasms ; enzymology ; genetics ; pathology ; RNA, Messenger ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Transfection

BACKGROUNDEndometrial carcinoma is one of the most common female tract genital malignant tumors. Nifedipine, an L-type calcium channel antagonist can inhibit cell proliferation of carcinomas. Recent studies indicated that a rise in the free cytosolic calcium ([Ca(2+)](c)) was a potent inducer of autophagy. Here, we investigated the relationship between nifedipine and autophagy in Hec-1A cells.

METHODSCells were cultured with nifedipine (10 µmol/L) and harvested at different times for counting cell number. MTT assay was applied to evaluate the cell viability and transwell assay to reveal cell migration. Apoptotic cells were detected with annexin V/PI assay. Then cells were treated with 3-methyladenine (3-MA) (2.5 mmol/L) for 0, 5, 15, 30, 60, and 120 minutes and the expression of the L-type calcium channel alpha1D (Cav1.3) protein was detected. At last, cells were cultured and assigned to four groups with different treatment: untreated (control group), 10 µmol/L nifedipine (N group), 2.5 mmol/L 3-MA (3-MA group), and 10 µmol/L nifedipine plus 2.5 mmol/L 3-MA (N+3MA group). Autophagy was detected with GFP-LC3 modulation by fluorescent microscopy, and expression of the autophagy-associated proteins (LC3, Beclin1 and P70s6K) by Western blotting and monodansylcadaverine (MDC) labeled visualization.

RESULTSProliferation of Hec-1A cells was obviously suppressed by nifedipine compared with that of the untreated cells for 24, 48, and 96 hours (P = 0.000 for each day). The suppression of migration ability of the nifedipine-treated cells (94.0 ± 8.2) was significantly different from that of the untreated cells (160.00 ± 9.50, P = 0.021). The level of early period cell apoptosis induced by nifedipine was (2.21 ± 0.19)%, which was (2.90 ± 0.13)% in control group (P = 0.052), whereas the late period apoptosis level reached (10.38 ± 0.96)% and (4.40 ± 0.60)% (P = 0.020), respectively. The 3-MA group induced a slight increase in the Cav1.3 levels within 15 minutes, but significantly attenuated the Cav1.3 levels after 30 minutes. There were more autophagic vacuoles labeled by MDC in the N group (20.63 ± 3.36) than the control group (6.29 ± 0.16, P = 0.015). GFP-LC3 localization revealed that the LC3 levels of cells in 3-MA group, N+3MA group, 3-MA group were 2.80 ± 0.29, 2.30 ± 0.17, and 1.80 ± 0.21, respectively. Cells in the N group showed significant augmentation of autophagy (P < 0.05). Western blotting analysis confirmed the down-regulation of LC3 levels in 3-MA group (0.85 ± 0.21) and N+3MA group (1.21 ± 0.12) compared with nifedipine treatment (2.64 ± 0.15, P < 0.05). The annexin-V-FITC/PI assay showed that the level of early period cell apoptosis induced in the N+3-MA group ((11.22 ± 0.91)%) differed significantly from that of the control group ((2.51 ± 0.70)%) and N group ((3.47 ± 0.39)%). Similarly, the late period level of the N+3-MA group ((55.19 ± 2.51)%) differed significantly from that of the control group ((15.81 ± 1.36)%) and the N group ((22.09 ± 2.48)%, P < 0.05). The down-regulated expression of P70s6k and up-regulated expression of the Beclin1 revealed significant differences between the N+3-MA group and control group (P = 0.025; Beclin1: P = 0.015).

CONCLUSIONSProliferation and migration in vitro of endometrial carcinoma Hec-1A cells are significantly suppressed by nifedipine. The nifedipine leads autophagy to oppose Hec-1A cells apoptosis. Autophagy inhibition by 3-MA leads down-regulation of Cav1.3 and enhances nifedipine-induced cell death. The nifedipine-induced autophagy is linked to Beclin1 and mTOR pathways.

Adenine ; analogs & derivatives ; pharmacology ; Apoptosis Regulatory Proteins ; physiology ; Autophagy ; drug effects ; Beclin-1 ; Calcium ; metabolism ; Calcium Channel Blockers ; pharmacology ; Calcium Channels, L-Type ; physiology ; Cell Line, Tumor ; Endometrial Neoplasms ; drug therapy ; pathology ; Female ; Humans ; Membrane Proteins ; physiology ; Nifedipine ; pharmacology ; Signal Transduction ; physiology ; TOR Serine-Threonine Kinases ; physiology

OBJECTIVETo search mutations in GLA gene in two Chinese families with classic Fabry disease.

METHODSTwo families with Fabry disease confirmed by pathological and clinical studies were reported here. In pedigree 1, 12 family members had paroxysmal pain on limb extremities. In pedigree 2, there were 8 patients and most of them had multi-organ involvement at the end stage of the disease. Two probands from the two families together with several of their family members were searched for mutations in GLA gene. After extraction of genomic DNA from peripheral leukocytes, all of the 7 exons and their flanking introns were amplified by PCR and directly sequenced.

RESULTSBoth the proband 1 and proband 2 were identified to be hemizygotes of novel GLA missense mutations. G132T (TGG-->TGT) mutation in exon 1, resulting in the substitution of amino acid from tryptophan to cysteine (W44C), was detected in proband 1. G874C (GCT-->CCT) mutation in exon 6, resulting in the substitution of amino acid from alanine to praline (A292P), was detected in proband 2. Mothers of the 2 probands were heterozygotes carrying the same mutation as their sons.

CONCLUSIONWe report here 2 novel missense mutations in two Chinese families with classic Fabry disease. Different mutations in the same gene can result in phenotypes with significant deviation. Several female patients with the same clinical manifestations as male patients in the 2 families suggest that the X-linked dominant inheritance of the disease, possibly related to be the random X chromosome inactivation.

Adolescent ; Asian Continental Ancestry Group ; Base Sequence ; DNA Mutational Analysis ; Exons ; genetics ; Fabry Disease ; genetics ; Family Health ; Female ; Heterozygote ; Humans ; Male ; Molecular Sequence Data ; Mutation, Missense ; Pedigree ; Polymerase Chain Reaction ; alpha-Galactosidase ; genetics

Objective To evaluate the efficacy of ultrasound-guided quadratus lumborum block for analgesia after laparoscopic cholecystectomy. Methods Sixty American Society of Anesthesiologists physi-cal statusⅠorⅡ patients, aged 18-64 yr, with body mass index of 20-27 kg∕m2 , scheduled for elective laparoscopic cholecystectomy, were divided into 2 groups ( n=30 each) using a random number table meth-od: ultrasound-guided transversus abdominis plane block group ( group T) and ultrasound-guided quadratus lumborum block group (group Q). In T and Q groups, bilateral ultrasound-guided transversus abdominis plane block and quadratus lumborum block were performed after general anesthesia induction, respectively, with 0. 25％ ropivacaine 20 ml for each side. All the patients received patient-controlled intravenous analge-sia with morphine after surgery, and visual analoge scale scores were maintained≤3 in the two groups. The cumulative consumption of morphine was recorded at 2, 4, 6, 12, 24 and 48 h after surgery. The occur-rence of adverse reactions such as nausea, vomiting, pruritus, over-sedation and respiratory depression was observed and recorded in the analgesic period. The operation time for nerve blocks and nerve blocks-related complications such as bleeding, accidental puncture into the abdominal cavity or intestinal injuries were re-corded. Results Compared with group T, the cumulative consumption of morphine was significantly de-creased at 6, 12, 24 and 48 h after surgery in group Q ( P<0. 05) . There was no significant difference in the operation time for nerve blocks between the two groups ( P>0. 05 ) . There were no adverse reactions such as nausea, vomiting, pruritus, over-sedation and respiratory depression or nerve blocks-related compli-cations in the two groups. Conclusion Ultrasound-guided quadratus lumborum block provides better effica-cy than transversus abdominis plane block when used for analgesia after laparoscopic cholecystectomy.