Hepatocellular carcinoma (HCC) development is closely related to overexpression of tumor promoters or down-regulation of tumor suppressors.The mammalian sirtuin family was found to be a nicotinamide adenosine dinucleotide (NAD/NADH)-dependent histone deacetylase (HDAC),which implicated in the regulation of critical biological processes through deacetylation modifying on histone and nonhistone.SIRT1 can regulate metabolism,aging,inflammation and cancer progression.In particular,more and more evidence proves that SIRT1 can act as a tumor promot er in hepatocellular carcinoma through deacetylation on tumor suppressors.On the other hand,SIRT1 can strongly suppress metabolic syndrome-associated liver cancer in the mouse model.This review will discuss the expression of sirtuin family member in liver cancer and its clinical significance.

Objective To study effect of Shikonin on human cervical cancer Hela cell growth suppression in vitro and its mechanism. Methods MTT assay was used to examine the growth inhibition of Shikonin in Hela cells.And then, the measurement of both ROS Levels and Mitochondrial Membrane Potential (ΔΨm ) were performed to clarify the mechanism of antitumor in Hela cells by Shikonin.Results Shikonin significantly inhibited the growth of Hela cells in a concentration-dependent manner.Shikonin increased generation of en-dogenous reactive oxygen species ( ROS) and decreased mitochondrial membrane potential.Furthermore, anti-oxidants N-acetylcysteine ( NAC) could significantly reduce the antitumor activity of SK in Hela cells.Conclusion These results suggest that mitochondrial aerobic respiration shift and endogenous ROS augmentation contribute to the action of Shikonin against Hela cells.

Objective To confirm the vancomycin loaded gelatin/β-TCP composite porous scaffolds could be used as sustained-release system,and investigate its efficiency of eliminating infections and repairing bone defects for the treatment of infected bone defects in rabbit.Methods The biodegradable gelatin sponge containing different contents (0,10％,30％,50％) of β-tricalcium phosphate ceramic (β-TCP) was prepared for the controlled-release of vancomycin and labeled with G-0 TCP,G-10 TCP,G-30 TCP and G-50 TCP respectively.Examinations of scanning electron microscopy,porosity analyses and mechanical test were performed.The K-B method was used to investigate the controlled release of vancomycin.Chronic Methicillin-resistant Staphylococcus aureus osteomyelitis models of rabbit were established.After thorough debridement,the infected bone defects were treated in four different groups:blank control group,G-0 TCP group,G-10 TCP group,and G-30 TCP group.At 4 and 8 weeks after implantation,X-ray and histological examinations were carried out to investigate the efficiency of eliminating infections and repairing bone defects.Results The prepared gelatin/β-TCP scaffold exhibited a homogeneously interconnected-3-D porous structure.And the β-TCP granules were localized evenly on the walls of the composite scaffold.There were no significant differences in the pore size of different scaffolds.However,the β-TCP granules can improve the interconnection.The porosity exhibited an obvious increase in G-10 TCP and G-30 TCP composite scaffolds compared with G-0 TCP scaffold.In contrast,too high content of β-TCP granules decreased the porosity.And the porosity exhibited an obvious reduction in the G-50 TCP composite scaffold.The compressive modulus of the vancomycin loaded scaffolds increased with the increase of the β-TCP amount.The scaffold G-0 TCP exhibited the longest duration of vancomycin release and the duration reached 8 weeks.With the increased content of β-TCP granules,the release duration shortened obviously.Compared with the G-50 TCP composite scaffold,the G-10 TCP and G-30 TCP composite scaffolds revealed a better controlled release of the drugs,and the total amount of the drugs was released within 7 weeks.However,the total amount of vancomycin released from the G-50 TCP composite scaffold lasted for 3 weeks.In the treatment of chronic MRSA osteomyelitis of rabbits,the G-30 TCP composite scaffold showed a better performance in the eliminating infections and bone defects repair.At 8 weeks after implantation,signs of osteomyelitis,including osteolysis,development of periosteal reactions,and sequestral bone formation were observed in the animals of blank control group.Signs of infection were absent in other treatment group.In the group treated with G-30 TCP composite scaffold,the bonedefects were repaired completely at 8 weeks after implantation.However,in the groups treated with G-0 TCP and G-10 TCP composite scaffold,the bone defects were not repaired.Conclusion The composite scaffolds could achieve local therapeutic drug levels over an extended duration.And the gelatin with 30％ β-TCP granules composite scaffold had optimal porosity,interconnection,mechanical properties and controlled release performances.It exhibited good performances in infection control and bone defect repair in the chronic MRSA osteomyelitis model.

In order to investigate in greater detail the two methods based on Hertz model for analyzing force-distance curve obtained by atomic force microscopy, we acquired the force-distance curves of Hela and MCF-7 cells by atomic force microscopy (AFM) indentation in this study. After the determination of contact point, Young's modulus in different indentation depth were calculated with two analysis methods of "two point" and "slope fitting". The results showed that the Young's modulus of Hela cell was higher than that of MCF-7 cell,which is in accordance with the F-actin distribution of the two types of cell. We found that the Young's modulus of the cells was decreased with increasing indentation depth and the curve trends by "slope fitting". This indicated that the "slope fitting" method could reduce the error caused by the miscalculation of contact point. The purpose of this study was to provide a guidance for researcher to choose an appropriate method for analyzing AFM indentation force-distance curve.
Actins ; Elastic Modulus ; HeLa Cells ; cytology ; Humans ; MCF-7 Cells ; cytology ; Microscopy, Atomic Force

10.3969/j.issn.2095-4344.2013.25.004

Objective To assess the association between NOD2 gene single nucleotide polymorphisms (SNPs) and leprosy in Chinese Yi population.Methods Whole blood samples were obtained from 300 patients with leprosy and 300 healthy human controls of Yi nationality in Sichuan province.Genomic DNA was extracted,and a SNaPshot assay was performed to determine the genotypes of four single nucleotide polymorphisms (SNPs) of the NOD2 gene,including rs9302752,rs7194886,rs8057341 and rs3135499.Chi-square test was conducted to compare allele frequency,and Hardy-Weinberg equilibrium was tested.Results The genotype distribution of all the four SNPs was consistent with Hardy-Weinberg equilibrium (all P ＞ 0.05).Significant differences were observed between the patients with leprosy and healthy controls in both genotype distribution and allele frequency of the SNP rs3135499 (both P ＜ 0.01),but not in those of the other three SNPs (all P ＞ 0.05).Conclusion The SNP rs3135499 of the NOD2 gene may be associated with the development of leprosy in Chinese Yi population.

Objective To construct the bait expression plasmid pGBKT7-GR of glucocorticoid receptor(GR)binding domain.Methods The fragments of GR binding domain was amplified by RT-PCR,and then was cloned into pMD18-T.After being verified by sequencing,it was subcloned into the bait expression vector pGBKT7.Then the bait vector pGBKT7-GR was transformed into AH109 yeast cells and the expression of the bait protein was analyzed by Western blot.Toxicity and self-activation of the bait protein were detected.Results GR binding domain was amplified and cloned into pMD18-T and pGBKT7 successfully.The bait vector was transformed into AH109 yeast cells successfully,without toxicity or self-activation.The expression of the bait protein was confirmed by Western blot.Conclusion The successful construction of bait expression vector of glucocorticoid receptor binding domain lays the foundation for constructing small molecule ligand yeast three-hybrid system.

Objective To construct microRNA (miRNA) expression vector targeting surviving,and to investigate its effect on transfected human colorectal carcinoma (HT-29) cell apoptosis and proliferation.Methods miRNA targeting survivin was synthesized and transfected HT-29 cells by lipofectin.HT-29 cells were cultured in the 6 orifices.The cultured cells were divided into control,liposome,negative control and positive control groups.Transient transfected cells were collected and the proliferation index and apoptosis rate of HT-29 cells were detected by flow cytometry.The expressions of survivin mRNA and protein were detected by RT-PCR and Western blot.Results The proliferation index and apoptosis rate of the positive control group were significantly higher compared with normal group,transfection group and mock-vehicle group (17.98％ ± 2.35％ vs 38.04％ ±2.11％ vs 36.73％ ±2.51％ vs 36.57％ ±3.05％; t =20.05,P＜0.01; t =18.75,P＜0.01; t=18.59,P＜0.01; 19.54％ ±1.74％ vs 3.13％ ±0.29％ vs 3.70％ ±0.44％ vs 3.61％ ± 0.50％ ; t =16.40,P ＜ 0.01 ; t =15.84,P ＜ 0.01 ; t =15.92,P ＜ 0.01).Survivin mRNA and protein expression levels were specifically suppressed in transfected HT-29 cells (t =0.68,P ＜0.01 ; t =0.58,P ＜ 0.01; t=0.61,P＜0.01;t=0.64,P＜0.01; t=0.62,P＜0.01;t=0.67,P＜0.01).Conclusion Survivin targeted silence can effectively decrease the expression of survivin mRNA and protein,induce colorectal carcinoma HT-29 cell apoptosis and inhibit cell proliferation.

Objective To investigate the feasibility of NR2B small interference RNA(NR2B siRNA)carried by water-soluble lipopolymer(WSLP)for treatment of neuropathic pain in rats.Methods One hundred healthy male SD rats weighing 180-200 g were randomly divided into 5 groups(n=20 each):normal control group (group C),sham operation group(group S),neuropathic pain group(group NP),group WSLP-NR2B siRNA (group W)and group WSLP-negative NR2B siRNA(group WN).Neuropathic pain was induced by partial ligation of sciatic nenre.WSLP-NR2B siRNA complex was formed by binding WSLP and NR2B siRNA.Normal saline.WSLP-NR2B siRNA complex and WSLP-negative NR2B siRNA 20μl were injected intrathecally after operation in NP,W and WN groups respectively.Mechanical withdrawal threshold(MWT)and thermal withdrawal duration (TWD)were measured before(baseline)and at 3,7,14 and 21 days after operation.Ten animals in each group were sacrificed on the 3rd day after operation and the lumbar segment(L4-6)of the dorsal root ganglia was removed for determination of the expression of NR2B mRNA and protein using RT-PCR and Western blot analysis.Results Sciatic nerve ligation significantly decreased MWT and prolonged TWD and increased NR2B mRNA and protein expression in group NP as compared with group C.WSLP-NR2B siRNA complex significantly reduced sciatic nerve ligation-induced hyperalgesia and decreased NR2B mRNA and protein expression in group W as compared with group NP.Conclusion WSLP not only mediates NR2B siRNA successfully and inhibits the expression of NR2B,but also reduces neuropathic pain in rats.

The aim of the present study was to establish a minimally invasive H reflex model in mice for the benefit of the research of clinical spinal cord injury and related diseases. Minimally invasive surgery was performed in hind limb of Kunming mouse under light anesthesia. The skin was incised at the point of one-third of the distance from greater trochanter to the base of the cauda. A pair of fine copper conductors were inserted into the shallow muscle using a syringe needle. After the needles were withdrawed, the retained conductors were ligated and fixed with the tissues surrounding the sciatic nerve as the first pair of stimulating electrodes. Another pair of conductors were inserted and fixed in medial malleolus close to the tibial nerve as the second stimulating electrodes. Copper conductor was inserted passing the skin above the proximal end of the metatarsal and fixed as the recording electrode. The reference electrode was placed at the walking pad in the base of the big toe using the same method. Electromyography (EMG) was used to record M and H waves in planta pedis muscles. The stimulus was a square wave with a width of 0.2 ms and frequency of 0.3 Hz. The latency time of the M and H waves which were induced from the two pairs of stimulating electrodes was recorded. Nerve conduction velocity (NCV) was then calculated from the distance between the cathodes of the stimulating electrodes and the latency time difference of M or H waves. The result showed the achievement ratios of H reflex induction were 92.73% and 81.82% in sciatic and tibial nerves, respectively. The latency time of H wave was about 7~10 ms. Motor nerve conduction velocity (MNCV) obtained was (25.84 ± 4.70) m/s (n = 35), while sensory nerve conduction velocity (SNCV) was (31.45 ± 7.30) m/s (n = 35). The method established in the present paper is simple to practice, does slight harm to the animal, and can produce waveforms with little interference. With these advantages, the method can be applied for the study of the latency of H reflex, and it is suitable for the researches which demands good physical condition of experimental animal during H reflex study. This model can also be applied to the detection of SNCV and MNCV.
Animals ; Electromyography ; H-Reflex ; physiology ; Hindlimb ; innervation ; Mice ; Neural Conduction ; physiology ; Tibial Nerve ; physiology