[Arthroscopic minimally invasive treatment of tibial intercondylar eminence fractures in children].

OBJECTIVE: To analyze the characteristics of children tibial intercondylar eminence fractures, and introduce arthroscopic minimally invasive techniques for the treatment of tibial intercondylar eminence fractures in children. METHODS: From January 2004 to December 2008, 12 children with tibial intercondylar eminence fractures were treated with cross Kirschner wire fixation after arthroscopic reduction. According to Meyers-McKeever classification systems, there were 1 case of type I, 4 cases of type II, and 7 cases of type III. There were 10 fresh and 2 old fractures in all. Among the patients, 10 patients were boy and 2 patients were girl,ranging in age from 8 to 13 years, with an average of 10 years. All the patients underwent arthroscopic exploration, reduction and fixation. During follow-up ranging from 10 to 36 months, the union of fracture, range of motion and stabilization of the knee were assessed. One patient was combined with lesions of the menisci, 1 patient with femoral trochlea cartilage injury, and 5 patients with meniscal entrapment under the bone. RESULTS: The heeling time averaged 5 weeks. No knee laxity or instability and no intercondylar notch impingement was detected in all cases at 3 months postoperatively. At same time, full range of motion of the affected knee returned, and the average Lysholm knee score was (92.7 +/- 2.5), the average Lysholm knee score was (96.4 +/- 1.7) at 6 months postoperatively. The Lachman test and ADT test was negative. CONCLUSION: The type II and type III tibial intercondylar eminence fractures occur frequently in children. Lesions of the menisci and cartilage occur seldom. The method of arthroscopic cross Kirschner wire fixation for the treatment of tibial intercondylar eminence fracture is easy to operate. Simultaneously, this technique is less invasive and allows early recovery. Also it coincidences with the characteristic rapid bone growth of children.

[Curcumin promotes apoptosis of esophageal squamous carcinoma cell lines through inhibition of NF-kappaB signaling pathway].

BACKGROUND & OBJECTIVE: Activation of NF-kappaB signaling pathway plays a critical role in the initiation and progression of carcinogenesis. However, the role of NF-kappaB pathway in esophageal squamous cell carcinoma (ESCC) has not been fully elucidated. Studies have shown that curcumin possesses anti-infection and anti-oxidation effects. This study was to evaluate whether curcumin could induce apoptosis through inhibition of NF-kappaB signaling pathway in ESCC cells. METHODS: Expressions of pIkappaBalpha and Bcl-2 were detected using Western blott after incubation of ESCC cells with curcumin (50 micromol/L) at different time points. Apoptosis and the number of viable ESCC cells were analyzed using flow cytometry and MTT, respectively, after the treatment of curcumin, 5-FU, or the combination of curcumin and 5-FU. RESULTS: In two ESCC cell lines, EC9706 and Eca109,curcumin inhibited IkappaBalpha phosphorylation and Bcl-2 in a time-dependent manner; curcumin alone increased cell apoptosis (P<0.05), and the effect became more prominent when it was combined with 5-FU (P<0.05); curcumin plus 5-FU exerted a stronger inhibition effect on cell proliferation than curcumin alone (P<0.05) or 5-FU alone (P<0.05). CONCLUSION: Curcumin inhibits the phosphorylation of IkappaBalpha, leading to suppression of proliferation, induction of apoptosis and an increase of the sensitivity of ESCC cell lines towards 5-FU.

[Effects of FMS-like tyrosine kinase 3 targeted RNA interference on proliferation and apoptosis of acute monocytic leukemia cell line THP-1].

OBJECTIVE: FMS-like tyrosine kinase 3 (FLT3) is a receptor tyrosine kinase that is constitutively activated in (70-90)% pediatric patients with acute myeloid leukemia (AML) and appears to confer an adverse prognosis. Although several FLT3-selective small molecule inhibitors and antibodies were developed with varied degrees of success, to address the specificity and resistance, new approaches for specifically targeted FLT3 are needed and RNA interference is a promising choice. The aim of the present study was to investigate the efficacy of suppression of FLT3 induced by small hairpin interfering RNA (shRNA) on myeloproliferation and apoptosis in an acute monocytic leukemia (AMOL) cell line THP-1. METHODS: FLT3-targeted small hairpin interfering RNA (FLT3-shRNA) was designed and synthesized by transcription system in vitro was transfected into THP-1 cells. Firstly FLT3 mRNA level was detected by semi-quantitative RT-PCR and FLT3 protein level was detected by flow cytometry (FCM) to verify the efficacy on FLT3-shRNA interference at 48 h after transfection. Cell growth viability was measured at 24 h, 48 h and 72 h after treatment with CCK-8. The distribution of cell cycle was assayed by FCM, and apoptosis was analyzed by DNA Ladder and Annexin V-FITC Staining at 48 h. RESULTS: FLT3 targeted shRNAs was synthesized successfully and the concentration of 15 nmol/L for 48 h could obtain desirable downregulation of FLT3 expression, the inhibitory percentages of FLT3 mRNA and protein were (72.95 +/- 2.07)% and (65.39 +/- 5.57)%, respectively. The suppression of FLT3 induced by FLT3-shRNA resulted in marked inhibition of cell growth and the inhibitory percentages were (36.66 +/- 3.67)% at 48 h, (35.56 +/- 0.73)% at 72 h. FLT3-shRNA induced the inhibition of cell cycle from G(0)/G(1) phase to S phase, the percentage of sub-G(0)/G(1) phase (65.71 +/- 4.47)% was higher than those in the PBS-control group (52.23 +/- 2.98)%, NC-shRNA control group (51.81 +/- 1.44)%, P < 0.01; the percentage of S phase (25.11 +/- 2.70)% was lower than those in the PBS-control group (34.41 +/- 4.07)% and NC-shRNA control group (32.50 +/- 1.46)%, P < 0.05. Furthermore treatment with FLT3-shRNA for 48 h resulted in clear apoptosis ladder, the percentage of early apoptosis detected by Annexin V-FITC was (18.59 +/- 2.07)% which was significantly higher than that in the PBS-control group (4.00 +/- 0.50)% and the NC-shRNA control group (6.06 +/- 0.70)%, P < 0.001. CONCLUSION: The suppression of FLT3 induced by the shRNA can effectively inhibit cell proliferation, and apoptosis induction on THP-1 cells, which indicates that this approach may bear the therapeutic potential on childhood AMOL.

[Expression of Notch1 gene in esophageal squamous cell carcinoma cell line EC9706 and its effect on cell apoptosis].

BACKGROUND & OBJECTIVE: Notch1 signaling pathway is closely associated with carcinogenesis and plays a key role in cell growth, proliferation, differentiation, and apoptosis. This study was to investigate the expression of Notch1 gene in esophageal squamous cell carcinoma (ESCC) cell line EC9706 and its effects on cell apoptosis. METHODS: The expression of Notch1 gene in EC9706 cells was detected by immunocytochemistry. Notch1 gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR). A eukaryotic expression vector pcDNA3.1-Notch1 (termed pcNICD) was constructed and transfected into EC9706 cells; pcDNA3.1 was also transfected as control. After transfection, the expression of Notch1 in EC9706 cells was detected by RT-PCR and Western blot, cell apoptosis was measured by flow cytometry (FCM). RESULTS: The expression of Notch1 gene was detected in EC9706 cells. The mRNA and protein expression of Notch1 gene in pcNICD-transfected EC9706 cells were significantly increased by about 3 folds of those in untreated and pcDNA3.1-transfected cells (P<0.01). However, there was no difference in Notch1 expression between untreated and pcDNA3.1-transfected EC9706 cells (P>0.05). The apoptosis rate was significantly higher in pcNICD-transfected EC9706 cells than in untreated and pcDNA3.1-transfected cells (P<0.01); there was no prominent difference between untreated and pcDNA3.1-transfected EC9706 cells (P>0.05). CONCLUSION: The activated Notch1 signaling pathway gives rise to the apoptosis of EC9706 cells, suggesting that Notch1 gene may be a new therapeutic target for ESCC.

[TAp63gamma-induced apoptosis mediated by apoptosis inducing factor in human esophageal squamous carcinoma EC9706 cells].

OBJECTIVE: To study the molecular mechanism of TAp63gamma-induced cell apoptosis. METHODS: Transcription and protein expression of apoptosis inducing factor and p63 were investigated by immunohistochemistry and RT-PCR in human esophageal squamous carcinoma cell line EC9706 respectively. Twenty-four hours after transfection with pcDNA3.1-TAp63gamma, the apoptosis and translocation of apoptosis inducing factor in EC9706 cells were studied by flow cytometry, laser confocal microscopy and mitochondrial/cytosol/nuclear extraction analysis respectively. Down-regulation of apoptosis inducing factor protein was achieved by RNAi and pretreatment with caspase inhibitor zVAD.fmk of EC9706 cells. RESULTS: Presence of protein expressions of apoptosis inducing factor and absence of TAp63gamma was observed in the cytoplasm of untransfected cells. RT-PCR verified the subtype of p63 in EC9706 cells was DeltaNp63. After 24 hours of transfection, both nuclear and cytoplasmic expression of apoptosis inducing factor protein were observed in cells transfected with TAp63gamma and p53 expression vectors, but not in cells transfected with control vector. Cell apoptosis rates were 1.37%, 13.64%, 4.52%, 4.03% and 1.91% in the pcDNA3.1 transfection group, pcDNA3.1-TAp63gamma transfection group, apoptosis inducing factor siRNA and pcDNA3.1-TAp63gamma transfection group, zVAD.fmk treatment group, and the group receiving apoptosis inducing factor siRNA, plus zVAD.fmk treatment and pcDNA3.1-TAp63gamma transfection, respectively. CONCLUSIONS: Apoptosis inducing factor of EC9706 cells is released from mitochondria into both the cytoplasm and nucleus during TAp63gamma induced apoptosis. Down-regulation of apoptosis inducing factor inhibits TAp63gamma-induced apoptosis. Overall, TAp63gamma-induced apoptosis is dependent on the expression of apoptosis inducing factor and caspase.

[Cloning and characterization of Hsp70a cDNA fragment of Dunaliella salina].

The present study is to obtain heat shock protein 70a cDNA fragment from Dunaliella salina. Two pairs of degenerate primers were designed according to conserved motifs of DIDLGTT,DQGNRTTP,PAYFNDS and ATKDAG of the homologous amino acid sequences and used to amplify hsp70a cDNA fragment from heat-shock-treated Dunaliella salina by nest PCR technique. The resulting PCR products were inserted into T-vector then transformed into JM109. Ten colonies were selected to determine their sequences. Homologous analysis of the deduced amino acid sequences were performed by BLAST and subsequently compared with GenBank data. Three of ten nucleotide sequences were obtained,of which there was 372 bp coding 126 amino acids. The sequences shared high homology with hsp70a,with identity 96% to Chlamydomonas reinhardtii, 94% to Petunia, 93% to Pisumsativum, 92% to tomato, 92% to human,90% to Drosophila and 89% to yeast respectively. It can be concluded that the cloned sequence is putatively hsp70a cDNA fragment from Dunaliella salina.