BACKGROUND: The study of isolation, purification, culture, cell labeling, inducing factors, effects of gene transfection on cytobiology, cell carrier construction, and time window for back transplantation of cell compound pertaining to marrow stromal cells (MSCs) is still in its infancy. OBJECTIVE: To search for an in vitro culture method that can be simply and effectively obtained. DESIGN, TIME AND SETTING: The present cytological in vitro experiment was performed at the Beijing Institute of Genome, Chinese Academy of Sciences between June 2006 and July 2007. MATERIALS: Eight specific pathogen-free New Zealand rabbits, aged 6 weeks, were provided by the Laboratory Animal Center, Institute of Genetics and Development, Chinese Academy of Sciences. METHODS: Under sterile condition, 1 mL rabbit bone marrow was taken and diluted with D-Hanks solution. Following centrifugation and subsequent supernatant removal, bone marrow was re-suspended using dulbecco's modified eagle's medium (DMEM) for single cell suspension. Next, single cell suspension was dropped onto the liquid surface of equal-volume lymphocyte separation medium (density: 1.077). Subsequent to centrifugation, cloudlike mononuclear cell layer was collected and re-suspended with DMEM containing 20% fetal bovine serum. The cells were inoculated at lxl0/cm2 and purified by adherent method. When 70%-80% of flask bottom was covered, cell digestion and passage was performed.MAIN OUTCOME MEASURES: Cell growth was observed with an operating microscope. Surviving cells were counted by Trypan blue viability test. Cell identification was performed by hematoxylin-eosin staining. Through the use of 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay, cell viability was detected to observe the cellular resuscitation of the cultured cells following cryopreservation. RESULTS: Twenty-four hours after inoculation, cells began to adhere to the wall, exhibiting short shuttle- or triangle-shaped appearance with different sizes of cellular processes. Three days later, adherent cells began to divide, and cell clusters could be found in some areas; One week later, most of cells exhibited scattered fibroblast-like growth; After passage, cells were evenly distributed with long shuttle-shaped appearance and arranged orderly. Following 3 passages, there wound be 5×106-1×107 adherent cells in 1 mL MSC suspension. Approximately 90% of MSCs survived and identified as mononuclear cells. Cells vigorously grew at days 1-6 after inoculation and reached a peak level at day 8, followed by a viability decline. After 56 days of resuscitation, frozen cells displayed a good and rapid growth. CONCLUSION: Highly purified MSCs can be acquired by gradient centrifugation of lymphocyte separation medium. Enough seeded cells can be obtained by in vitro culture and the cellular viability does not change after frozen preservation and resuscitation.

BACKGROUND: SOX-9 plays an important role in occurrence and development of cartilaginous tissues,enhances agglutination of mesenchymal cells,has structural domains of transcriptional activation,and can directly regulate the transcription,OBJECTIVE: To construct pDC316-SOX-9 plasmid for transfection of rabbit bone marrow stromal cells (BMSCs) using SOX-9 gene,and to investigate the effects of SOX-9 gene on growth characteristics of BMSCs and product expression. DESIGN,TIME AND SETTING: The cell gene engineering in vitro experiment was performe,d at the Beijing Institute of Genome, Chinese Academy of Scienees from June 2006 to January 2007.MATERIALS: Eight SPF New Zealand rabbits aged 6 weeks were used for culture of BMSCs.METHODS: pDC316-SOX-9 plasmid was used for transfection of rabbit BMSCs by liposome mediated method,Cell transfection included a SOX-9 plasmid transfection group,a blank plasmid group and a blank control group (only treatment of liposome). MAIN OUTCOME MEASURES: Cell morphology; growth activity; The SOX-9 protein expression in rabbit BMSCs were detected by immunohistochemistry,hematoxylin-eosin staining,reserve transcriptase-polymcrase chain reaction (RT-PCR) and enzyme-labeled immunosorbent assay (ELISA). RESULTS: Some cell colonies were detected at 4 days after pDC316-SOX-9 plasmid transfection.Spindle-shaped cells were collected after clone amplification.Cells in the blank control group gradually died over time.Cell activities in the SOX-9 plasmid transfeetion group and the blank plasmid group significantly prolonged,reached a peak at 2 weeks of transfection,and then gradually decreased.At 6 days,BMSCs were yellow in the SOX-9 plasmid transfection group following immunohistochemistry,expressing SOX-9 protein.Hematoxyliu-eosin staining showed many dikaryocytes,rich cell proliferation,similar to chondroblasts.No SOX-9 protein expression and unproductive cell proliferation in the blank plasmid group.SOX-9 mRNA was detected in the SOX-9 plasmid transfection group,but SOX-9 mRNA was not detected in the blank plasmid group and blank control group.SOX-9 levels were significantly higher in the SOX-9 plasmid transfection group than in the blank plasmid group and blank control group at 24,48 and 72 hours,1,2 weeks (P＜ 0.01).CONCLUSION: Rabbit BMSCs were successfully transfected with pDC316-SOX-9 plasmid to enhance cell growth activity and to persistently stably secrete SOX-9 protein,resulting in the differentiation of BMSCs into cartilages.

Objective To investigate the anatomic feature and special clinical manifestations of variant right intrahepatic bile duct draining into left hepatic bile duct near the umbilical portion. Methods Variant right intrahepatic bile ducts joining into left hepatic bile ducts near the umbilical portions were identified through cholangiograms in 52 patients, who were included in this study. Their history, clinical process and operations were reviewed. Results There were total 38 cases of intrahepatic gallstone in this group. High incidence of intrahepatic calculi was found in variant right intrahepatic bile ducts (23/38 cases, 60.52%) and left hepatic ducts (33/38 cases, 86.84%). Most of these cases were accompanied with dilatation and stricture of bile ducts in these area. The gallstones in the variant right intrahepatic bile ducts were not detected in 8 cases (8/23) and the rate of residual gallstone was as high as 86.95%(20/23). Injury of variant right intrahepatic bile duct took place when left hepatectomy was performed in one case. Conclusion Gallstone is very likely to be formed in the variant right intrahepatic bile duct due to derangement of bile hydrokinetics and compression of blood vessel. Special attention should be paid to the diagnosis and operation of this abnormity.

Objective To compare three different internal fixations using dual plates in treatment of type C distal humerus fractures.Methods A total of 59 patients with type C distal humerus fractures fixed with dual plates between January 2004 and December 2008 were enrolled in the study,including 34 patients managed by perpendicular dual plate internal fixation (Group A),14 patients by dorsal dual plate internal fixation (Group B),and 11 patients by parallel dual plate internal fixation (Group C).Functional outcomes of injured elbow joints were assessed using Mayo elbow performance score (MEPS).Fisher' s exact probability,chi-square test and variance analysis were used to compare the perioperative variables among groups.Results The patients were followed up for average 28 months (range,12-55 months),which showed that all fractures were smoothly healed with satisfactory curative effects in each group.There were no significant differences with respect to functional outcomes among three groups.The patients with surgery difficulty in Group C were more than those in other two groups.Besides,an additional intercondylar screw was always required in Groups A and B,but rarely in Group C.Conclusions All internal fixations with three dual plates are effective in management of type C distal humeral fractures.The location of plates is determined by experiences of orthopedic surgeons and specific fracture type.

During the teaching activities, to stimulate students' subject awareness and encour-age them to play the main role in class activities are inevitable trends in the reform of college educa-tion. Students’ subjective activity is a key to the teaching effect of regional anatomy, a course mainly based on experimental program. Department of Human Anatomy in Chongqing Medical University lay-outs regional anatomy teaching program to develop the students' subjective activities in learning from the course specialty: in preview and review, to train students' image-thinking by drawing; in anatomi-cal operation link, to cultivate students' interest in learning and innovation by discussing the relation-ship of anatomical structure and clinical disease and identifying the variation of structure; in the eval-uation process, taking the formative evaluation system to promote the students' initiatives and ensure the objectiveness and fairness. The implementation of these measures promotes the regional anatomy teaching quality.

BACKGROUND:Hepatic fibrosis in chronic liver disease can be reversed. Studies have shown that Periplaneta americana extract has anti-fibrosis effect, and has protective effect on the experimental hepatic fibrosis rats.
OBJECTIVE:To observe the effect of Periplaneta americana extract sticky sugar amino acid on hepatic fibrosis, and to primarily explore the mechanism of sticky sugar amino acid against hepatic fibrosis.
METHODS:Rat models of immune hepatic fibrosis were induced by pig serum and intragastricaly administered 0.5, 0.25, 0.10 g/kg sticky sugar amino acid. Four indexes of hepatic fibrosis were detected by radioimmunoassay. Immunohistochemical staining wasused to measure transforming growth factor beta 1, tissue inhibitor of metaloproteinase protein expression intensity and positive cel rate, to determine the correlation of different concentrations of sticky sugar amino acid, transformation growth factorbeta 1 and tissue inhibitor of metaloproteinase.
RESULTS AND CONCLUSION:(1) The Periplaneta americana extract sticky sugar amino acid reduced the levels of laminin, type III procolagen, type IV colagen and hyaluronidase (P< 0.01) and reduced the expression of transformation growth factor beta 1 and tissue inhibitor of metaloproteinase 1 in liver tissue (P< 0.01). (2) Sticky sugar amino acid concentration and transformation growth factor beta 1 and tissue inhibitor of metaloproteinase 1 protein expression showed a significant negative correlation (|r| > 0.9). (3) Results confirmed that the Periplaneta americana extract sticky sugar amino acid can change the reversal of hepatic fibrosis. Its mechanism of action is associated with expression of transforming growth factor beta 1 and tissue inhibitor of metaloproteinase 1 inhibited by sticky sugar amino acids.

In the current study, a total of nineteen triterpenoids (1-19) from 60% EtOH extracts of Stauntonia obovatifoliola Hayata subsp. intermedia stems were separated and purified by solvent extraction and chromatographic methods including silica gel, ODS as well as preparative HPLC. According to the results of chemical reactions and spectral data, compounds were identified as: lupeol (1), betulinonic acid (2), betulinic acid (3), 3-epi-betulinic acid (4), quinatic acid (5), 24-O-acetyl quinatic acid (6), 3-O-α- L-arabinopyranosyl-30-nor-hederagenin-28-O-α-L-rhamnopyranosyl-(1 --> 4) -β-D-glucopyranosyl-(1 --> 6) -β-D-glucopyranosyl ester (7), Stauntoside A (8), kalopanax saponin A (9), kalopanax saponin J (10), Kizuta saponin K10 (11), 3-O-α-L-rhamnopyranosyl (1--> 2) -α-L-arabinopyranosyl-hederagenin-28-O-β-D-xylopyranosyl-(1 --> 6) -β-D-glucopyranosyl ester (12), kalopanax saponin B (13), 3-O-α-L-rhamnopyranosyl-(1 --> 2) -α-L-arabinopyranosyl-hederagenin-28-O-β-D-glucopyranosyl-(1 --> 6) -β-D-glucopyranosyl ester (14), sieboldianoside A (15), septemoside A (16), kalopanax saponin K (17), septemloside I (18), and 3-O-α-L-arabinopyranosyl (1 --> 2)-β-D-glucuronopyranosyl- hederagenin (19). Among them, compounds 4, 6, 10, 12, 14, and 16-19 were isolated from the Stauntonia genus for the first time, and compound 6 was a new natural product.
Drugs, Chinese Herbal ; chemistry ; Magnetic Resonance Spectroscopy ; Magnoliopsida ; chemistry ; Molecular Structure ; Plant Stems ; chemistry ; Spectrometry, Mass, Electrospray Ionization ; Triterpenes ; chemistry

Wuzhuyu Tang is a classical formula for treating migraine, but its' pharmacological ingredients is unclear yet. Present study employed the everted intestinal sac model to collect the absorption samples of 10 kinds of Wuzhuyu decoction, and then analyzed the contents of 9 ingredients in Wuzhuyu Tang and absorption samples quantitatively or semi-quantitatively by HPLC-DAD method. Reserpine was used to establish the mice model of migraine, and then the contents and activities of 5-hydroxytryptamine, noradrenaline, dopamine, nitric oxide and nitricoxide synthase in brain tissues and serums were determined respectively after oral administration of Wuzhuyu Tang. Using the partial least squares regression method to correlate the total absorption quantity of 9 ingredients and pharmacodynamics. The result shows that limocitrin-3-O-beta-D-glucoside, ginsenoside Rg1 and Rb1, rutaevine, limonin, evodiamine and rutaecarpine are the main ingredients influenced the effects in absorption samples in everted intestinal sacs, especially ginsenoside Rg1, rutaevine, evodiamine and rutaecarpine among them have obvious improving effects to most pharmacodynamics index, might be the pharmacological ingredients influenced the therapeutical effects of Wuzhuyu Tang treating migraine.
Animals ; Drugs, Chinese Herbal ; pharmacology ; Female ; Intestinal Absorption ; drug effects ; Intestines ; drug effects ; Male ; Mice ; Mice, Inbred ICR ; Migraine Disorders ; drug therapy ; Rats ; Rats, Wistar

OBJECTIVETo investigate the antitumor efficacy of death receptor 5, its ligand (TRAIL) and DR5mAb in human hepatocellular carcinoma.

METHODSExpression of DR5 in the HCC cell lines HepG2, SMMC 7721 and normal human liver cell line LO2 was measured at mRNA and protein level by semi-quantitative RT-PCR and Western blot, respectively. MTT method was used to measure the cell viability and flow cytometry assay was used to detect apoptosis so as to observe the inhibitory effect of TRAIL and DR5mAb on HCC cells.

RESULTSDeath receptor 5 was highly expressed in the HCC cell lines, but rarely expressed in normal human liver cell line (P < 0.01). With the increase of TRAIL concentration, the cell viability of HCC cells decreased gradually. However, when the concentration of TRAIL was above 1000 ng/ml, HCC cells were resistant to TRAIL, but still sensitive to DR5mAb. After incubation with DR5mAb (1000 ng/ml) for 24 h, the rate of apoptosis in HCC cells reached to 52.45% +/- 0.57%, which was higher than that incubated with TRAIL under the same condition (14.74% +/- 0.48%) (P < 0.05). The cell viability of normal human liver cell line treated with TRAIL tended to decline with the increase of the concentration, which was significantly different from that of matched control group. But DR5mAb had little effect on normal human liver cell line.

CONCLUSIONDeath receptor 5 as a target plays an important role in the course of HCC apoptosis induction. Agonistic monoclonal antibody specific for human DR5 can selectively and effectively kill hepatocellular carcinoma cells in vitro, while is not harmful to normal human hepatocytes. It reveals that DR5mAb might provide a new direction in hepatocellular carcinoma treatment research.

Antibodies, Monoclonal ; pharmacology ; Apoptosis ; drug effects ; Carcinoma, Hepatocellular ; metabolism ; pathology ; Cell Line ; Cell Line, Tumor ; Cell Survival ; drug effects ; Humans ; Liver Neoplasms ; metabolism ; pathology ; RNA, Messenger ; biosynthesis ; genetics ; Receptors, TNF-Related Apoptosis-Inducing Ligand ; biosynthesis ; genetics ; immunology ; TNF-Related Apoptosis-Inducing Ligand ; biosynthesis

Objective: To explore the association between reading and writing posture with the occurrence of myopia in Chinese children and adolescents, so as to provide a reference for reducing its prevalence among primary and secondary school students. Methods: Using data from the 2020 survey on the current status of hyperopia reserve in primary and secondary school students from six provinces/cities, namely Beijing, Zhejiang, Chongqing, Shaanxi, Liaoning, and Henan selected by multi stage stratified cluster probability sampling method during November 2020 to June 2022. A total 16 782 students who completed the optometry examination of cycloplegia according to the Children and Adolescents Myopia Screening Standard and questionnaire survey were included for analyzing analyze the association between reading and writing posture with myopia. Results: The overall detection rate of myopia among primary and secondary school students was 45.7%, of which 35.0% were primary school students, 84.1% junior high school students, and 90.4% senior high school students. Poor reading and writing posture was found in 73.6% of primary and secondary school students. Adjusting for confounding factors such as gender, school period, region, outdoor time every day, and whether parents were myopic, the results of the multilevel Logistic regression showed that the following factors were positively correlated with myopia:poor reading and writing posture ( OR=1.17, 95%CI =1.07-1.28), never/occasionally reading and writing with a reading distance which was more than one foot away between the eyes and book ( OR=1.28, 95%CI=1.08-1.53, OR=1.23, 95%CI = 1.08- 1.40), teachers occasionally reminding the child of their reading and writing posture ( OR=1.13, 95%CI =1.01-1.25), and often/always reading and writing while lying down or with the face on the arm ( OR=1.15, 95%CI=1.01-1.32, OR=1.46, 95% CI = 1.17-1.82), always reading and writing with the head in the hand ( OR=1.56, 95%CI =1.20-2.01). Further, a negative correlation was detected between myopia and parents occasionally reminding their children of their reading and writing posture ( OR= 0.85 , 95%CI =0.76-0.96) ( P <0.05). Conclusion Poor reading and writing posture is a risk factor for the development of myopia in primary and secondary school students, and interventions for reading and writing posture need to be strengthened to reduce the occurrence of myopia among primary and secondary school students.