Isolation, culture and identification of rabbit bone marrow-derived vascular endothelial progenitor cells / 中国实验血液学杂志

The aim of study was to set up a suitable method of isolation, culture and identification of endothelial progenitor cells (EPC) derived from rabbit bone marrow. Density gradient centrifugation was used to isolate mononuclear cells from bone marrow, the isolated mononuclear cells were cultured with specific culture medium for EPCs. EPCs were identified by cellular morphologic observation, immunohistochemistry testing, flow cytometry and the function test of taking up Dil-ac-LDL and FITC-UEA-1. The results indicated that the newly isolated bone marrow-derived mononuclear cells exhibited a round appearance, following culture for 48 hours, adherent cells grew in colony cluster, presenting with round or irregular appearance, and nuclear division was obvious. On day 7, flaky cell colonies mutually connected together, presenting with spindle-shaped cells. Immunohistochemistry testing in the EPCs showed CD133(+), CD34(+), VIII factor(++), KDR(++); flow cytometry testing showed that the positive rate of CD133 was (18.23+/-7.12)%, the positive rate of CD34 was 47.71+/-14.85%, the positive rate of CD31 was (71.61+/-13.51)%, the positive rate of KDR was (87.24+/-11.40)%. And more than 80% EPC could take up both Dil-acLDL and FITC-UEA-1. It is concluded that the mononuclear cells isolated from bone marrow by density gradient centrifugation can differentiate into EPCs under special culture situation.

Predictors of mortality in critically multiple trauma patients after damage control surgery / 中华外科杂志

<p><b>OBJECTIVE</b>To investigate the efficiency of damage control surgery (DCS) and predictors of mortality in critically multiple trauma patients.</p><p><b>METHODS</b>From May 1998 to February 2007, DCS were carried out in 27 patients with critically multiple trauma. Of the patients 15 cases survived (survival group) and 12 cases died (dead group). The surgical complications, causes of death, demographic, physiologic and medical parameters were collected and compared between the two groups. Multiple logistic regression analysis were performed to identify possible predictors of mortality.</p><p><b>RESULTS</b>The incidence of surgical complications was 37.0 percent, and the intra-abdominal infections was the most frequent (18.5%). The overall mortality rate was 44.4 percent. The most common causes of death was multiple organ dysfunction syndrome (50.0%). With respect to predicting mortality, statistically significant differences was found in parameters as age, injury severity score (ISS), initial temperature and base excess (BE), estimated blood loss, initial ICU temperature and length of hospital stay. Older age, increased absolute value of initial BE and lower initial ICU temperature were determined as independent predictors of mortality on multiple logistic regression analysis.</p><p><b>CONCLUSIONS</b>There is a comparable high morbidity and mortality rate in severely injured patients managed with DCS. Increased age, a larger absolute value of initial BE and lower initial ICU temperature could independently predict death of the patients.</p>

Value of dynamic subtraction technique of magnetic resonance imaging in preoperative TNM-staging assessment of gastric carcinoma / 中华胃肠外科杂志

<p><b>OBJECTIVE</b>To explore the value of dynamic subtraction technique of magnetic resonance imaging MRI in preoperative TNM-staging assessment of gastric carcinoma.</p><p><b>METHODS</b>MRI was performed in 39 patients with gastric carcinoma diagnosed by postoperative pathology.The results of MRI were prospectively analyzed by one professor and compared with the corresponding pathological findings.</p><p><b>RESULTS</b>In comparison with pathological results, the accuracy of MRI for T stage was 82.1%, for N stage was 71.8%, and for M stage was 84.6% respectively. The accuracy of MRI for TNM stage was 71.8%, which revealed concordance between the preoperative TNM-staging and postoperative pathological findings (Kappa= 0.671-0.763, P<0.05).</p><p><b>CONCLUSION</b>MRI plays an important role in the assessment of invasion depth of gastric carcinoma, lymph node and distant organ metastases,which has unique priority in preoperative TNM-staging assessment of gastric carcinoma.</p>

Effect of free hemoglobin on hemorrhagic shock in pigs: TNF-alpha, IL-6 expressions in serum and rates of MODS after the blood transfusion / 中国实验血液学杂志

This study was aimed to investigate the expressions of tumor necrosis factor-alpha (TNF-alpha), Interleukin-6 (IL-6) in serum and the incidence of multiple organ dysfunction syndrome (MODS) in pigs with hemorrhagic shock after the blood transfusion simultaneously combined with different doses of free hemoglobin (FHb) so as to provide guidance of banked blood with high concentration of FHb during war injury through understanding effect of FHb on the animals. The different doses of FHb were given intravenously during the recovery of pig from shock, the vital signs and functional changes of vital organs were monitored and the incidence of MODS was determined, as well as the serum specimens were collected and the TNF-alpha, IL-6 levels in serum were detected by ELISA. The results showed that there were statistical differences of serum levels of TNF-alpha and IL-6 in pigs after FHb 10 mg/kg infusion, as compared to shock control group. There was significantly difference of the serum levels of TNF-alpha, IL-6 after FHb 15 mg/kg infusion, compared to the control group. The incidence of MODS increased significantly. It is concluded that the blood infusion containing high dose (more than 10 mg/kg) of FHb influences significantly on the cytokines in pigs with hemorrhagic shock, and increases damage of cytokines to vital organs and the incidence of MODS. The tolerance dose of the pigs to free hemoglobin is about 10 mg/kg or so. The infusion of blood with less than 10 mg/kg is relatively safe for pig in hemorrhagic shock.

Clinical management of abdominal trauma / 中华创伤杂志(英文版)

<p><b>OBJECTIVE</b>To improve the prognosis of patients with abdominal trauma.</p><p><b>METHODS</b>Between January 1993 and December 2005, 415 patients were enrolled in this research. The patients consisted of 347 males and 68 females with mean age of 36 years (ranging from 3-82 years). All abdominal traumas consisted of closed traumas (360 cases, 86.7%) and open traumas (55 cases, 13.3%).</p><p><b>RESULTS</b>A total of 407 cases (98.1%) were fully recovered from trauma and the other 8 cases (1.9%) died of multiple injuries. The mean injury severity score (ISS) of all patients was 22 while the mean ISS of the patients who died in hospital was 42. Postoperative complications were seen in 9 patients such as infection of incisional wounds (6 cases), pancreatic fistula (2 cases) and intestinal fistula (1 case). All these postoperative complications were cured by the conservative treatment.</p><p><b>CONCLUSION</b>Careful case history inquisition and physical examination are the basic methods to diagnose abdominal trauma. Focused abdominal ultrasonography is always the initial imaging examination because it is non-invasive and can be performed repeatedly with high accuracy. The doctors should consider the severity of local injuries and the general status of patients during the assessment of abdominal trauma. The principle of treatment is to save lives at first, then to cure the injuries. Unnecessary laparotomy should be avoided to reduce additional surgical trauma.</p>

Anti-tumor effect of gene-viral therapeutic system CNHK300-murine endostatin on nude mouse gastric cancer / 中华胃肠外科杂志

<p><b>OBJECTIVE</b>To investigate the anti-tumor effect of a novel gene-viral therapeutic system CNHK300-murine endostatin (CNHK300-mE) on gastric cancer.</p><p><b>METHODS</b>SGC-7901 gastric cancer cells (5 x 10(7) cells/mouse) were injected s.c. into the right flank of Balb/c nude mice, grown to 4-5 mm to demonstrate tumor take, and 10(9) pfu/100 microl CNHK300-mE virus was injected into tumors. Tumor sizes were measured with calipers every other day. Serum samples were obtained by retro-orbital puncture and level of endostatin expression in serum was quantitated by ELISA. Fifteen days after treatment, all mice were sacrificed and tumors were excised for immunohistochemical staining of PCNA, hexon and vWF. Tumor cell apoptosis was detected by TUNEL method.</p><p><b>RESULTS</b>From the 7th day post-treatment, the bearing tumors of mice treated with CNHK300-mE were significantly smaller than those of control group treated with PBS. Seven days after treatment, expression of endostatin was (2115 +/- 770) ng/ml, significantly higher than that of control group. Immunohistochemical staining indicated that hexon was expressed in treated tumor cells, and PCNA LI (label index) [(55.0+/-1.4)% vs control (74.1 +/- 0.4)%, P<0.05], microvessel density (MVD) of CNHK300-mE treated tumors decreased significantly. Apoptosis obviously increased in tumor cells[(78.4 +/- 9.1)% vs control (15.2 +/- 0.5)%, P<0.01]. Apoptosis bodies and crystal grid were found in tumor cell nuclear by electron microscope.</p><p><b>CONCLUSIONS</b>Gene-viral therapeutic system CNHK300-murine endostatin can replicate in gastric cancer cells. The mouse endostatin gene cloned into CNHK300-mE expressed in high level. CNHK300-mE may induce tumor cells apoptosis, reduce the expression of PCNA and efficiently suppress gastric cancer growth through inhibiting tumor angiogenesis.</p>

Effect of replication- competent adenovirus-mediated interleukin- 12 gene to chemotherapeutic sensitivity on gastric cancer cell / 中华胃肠外科杂志

<p><b>OBJECTIVE</b>To investigate the effect of replication-competent adenovirus-mediated interleukin-12 gene to chemotherapeutic sensitivity on gastric cancer cell.</p><p><b>METHODS</b>Replication-competent adenovirus and replication-competent adenovirus- mediated interleukin- 12 gene was constructed and expanded separately. The mortality of gastric cancer cell caused by the CNHK200- mIL- 12, Onyx- 015 in combination with different dosages of chemotherapeutic agents were evaluated by MTT assay at the same viral titer with a series of different dosages of chemotherapeutic agent,or at a series of different viral titers with the same dosage of chemotherapeutic agent. The curative effect to the xenografts gastric tumor in nude mouse was also observed by two viruses solely or together with 5-Fu.</p><p><b>RESULTS</b>The lytic activity of replication-competent adenovirus to gastric cancer cell line SGC-7901 was relatively poor at MOI value of 0.5, but it could be improved significantly when combined with chemotherapeutic agents of ADM, 5-Fu or CAP compared to the simple chemical therapy (P< 0.05). Chemotherapeutic agent 5- Fu could not effectively kill SGC-7901 when used at a relatively low dosage of 10microg/ml,whereas its activity could be improved when combined with a replication-competent adenovirus,and the killing rate was much higher than that with replication-competent adenovirus solely (P< 0.05). The gastric tumor xenografts was prevented and killed by replication adenovirus solely or combined with 5-Fu.</p><p><b>CONCLUSION</b>The replication- competent adenovirus- mediated interleukin- 12 gene can increase the chemotherapeutic sensitivity on gastric cancer cell. There is synergetic effect between the replication adenovirus and the chemotherapeutic agents in killing gastric cancer cell.</p>

Potent antitumoral effects of a novel gene-viral therapeutic system CNHK300-mEndostatin in hepatocellular carcinoma / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>The expression of therapeutic gene and its anti-tumor effects will be augmented and a synergism of oncolytic virus with the therapeutic gene is speculated. This study was undertaken to assess the anti-tumor effects of a novel gene-viral therapeutic system CNHK300-mEndostatin (CNHK300-mE) in hepatocellular carcinoma (HCC).</p><p><b>METHODS</b>A novel gene-viral therapeutic system named CNHK300-mE was constructed using the human telomerase reverse transcriptase (hTERT) promoter to drive the expression of the adenovirus E1A gene and cloning the therapeutic gene mouse endostatin into the adenovirus genome. By the tissue culture infectious dose 50 (TCID50) method and cytoviability assay, the replicative and cytolytic capabilities of CNHK300-mE in two HCC lines (HepGII and Hep3B) and one normal cell line (MRC-5) were analyzed, and the transgene expressions of mouse endostatin in vitro and in vivo were detected by Western blotting and ELISA assay. Tumor growth suppression and anti-angiogenesis effects in vivo were investigated using nude mice xenografts model derived from SMMC-7721 HCC cells.</p><p><b>RESULTS</b>The 3296-fold replicating capacity of CNHK300-mE in HCC cell lines versus in the normal cell line at 96 hours post infection and the 25-fold effective dose for killing 50% cells (ED50) in the normal cell line versus HCC cell lines, which were both superior to ONYX-015, were observed. Tumor growth suppression of CNHK300-mE superior to either Ad-mE or ONYX-015 was demonstrated (P < 0.01) and the anti-angiogenic effects in vivo superior to Ad-mE were also observed with immunohistochemical staining of von Willebrand factor. In comparison with non-replicative adenovirus Ad-mE, the transgene expression of mE mediated by CNHK300-mE was significantly higher in vitro (P < 0.005) and in vivo (P < 0.05).</p><p><b>CONCLUSION</b>Being capable of replicating in and lysing the telomerase-positive HCC cells and mediating effective expression of the therapeutic gene in vitro and in vivo, the novel gene-viral therapeutic system CNHK300-mE is potentially effective in the treatment of HCC.</p>

Expression and significance of major histocompatibility complex classⅡgene in multiple organ dysfunction syndrome / 中华创伤杂志

Objective To investigate the expression and significance of major histocompatibility complex classⅡgene in multiple organ dysfunction syndrome.Methods Two-hit porcine model of MODS was duplicated in 18 swine that were randomly assigned into experimental group(Group M,n=9) and control group(Group C,n=9).The Group M was given compound factors including hemorrhagic shock,reperfusion injury and endotoxemia,and the Group C only underwent anesthesia and arterious/ve- nous eannula.After seven days,the animals were killed to remove splenic tissues fro extracting total RNA by Trizol method.The primer of SLA-DQA(MHC classⅡgene of swine)was designed to construct cD- NA by reverse transcription and the quantity of SLA-DQA mRNA detected with real time fluorescent quan- titative polymerase chain reaction(real time FQ-PCR).The standard curve was described by UVP com- puter image analysis system.Results The mortality of Group M was 78%(7/9),and the incidence rate of MODS was 89%(8/9).The expressing quantity of Group M was(1.376?1.006)?10~3,signifi- cantly lower than(5.330?3.053)?10~3 of Group C(P＜0.01).Conclusion Duplication of por- cine MODS model is satisfactory.Down-regulation of MHC classⅡgene may be due to control of classⅡtransactivator(CⅡTA)and release of multiple eytokine,such as TNF-?and IL-10.

Changes of Th1/Th2 balance in multiple organ dysfunction syndrome / 第二军医大学学报

Objective:To explore the pathogenesis of multiple organ dysfunction syndrome(MODS)with respect to the bal- ance of Th1/Th2.Methods:Eighteen healthy male minipigs,weighing 22-30 kg,were randomly divided into two groups: MODS group and control group.Double-hit method including hemorrhagic shock and endotoxiemia was used to establish the porcine MODS model.The peripheral vein blood samples were collected at different time-points(before bloodletting,before en- dotoxin injection,1 h,24 h,48 h and 72 h after endotoxin injection)in the two groups.The spleen samples were collected after death of the animals.Plasma levels of IFN-?and IL-4 were detected by enzyme-linked immunosorbent assay(ELISA).Real- time PCR was used to detect the expression of IFN-?,IL-4,T-bet and GATA-3 mRNA(The latter 2 were the key transcription factors associated with Th1/Th2 response)in the spleen samples.Results:The plasma levels of IFN-?and IL-4 quickly reached the peak values 1 h after the endotoxin injection,then the level of IFN-?decreased quickly.The ratio of IFN-?/IL-4 was signifi- cantly lower than the baseline value 72 h after endotoxin injection(P=0.000).The ratio of IFN-?/IL-4 mRNA in MODS group was obviously lower than that in the control group(P=0.020);the ratio of T-bet/GATA-3 was also lower in MODS group (P=0.038).Conclusion:The shift from Th1 to Th2 occurs in the progress of MODS.