OBJECTIVENucleic vaccine of pVVP3IL-18HN expressing apoptin gene, Newcastle disease virus HN gene and IL-18 gene were constructed to observe the combinative antitumor effect of the above three genes.

METHODSEukaryotic expression plasmid pVVP3IL-18HN was constructed by inserting apoptin gene and fragment comprising fused IL-18HN gene and IRES promoter into the downstream of CMV promoter of vector pVAX1. The expression of inserted gene was identified by RT-PCR, indirect immunofluorescence and Western-blot. The recombinant plasmid was introduced into Hep-2 cells by liposome, then suppression rate of Hep-2 of different time and different quantity was calculated according to MTT results.

RESULTThe recombinant plasmid of pVVP3IL-18HN suppressed Hep-2 successfully and its suppression rate was up to 61.9% with 20 microg/ml, incubation of 72 hours.

CONCLUSIONThe nucleic vaccine constructed pVVP3IL-18HN had antitumor effect on Hep-2. It may can be used to the therapy and research of laryngeal carcinoma.

Cancer Vaccines ; biosynthesis ; Gene Expression ; Genetic Vectors ; HN Protein ; genetics ; Hep G2 Cells ; Humans ; Interleukin-18 ; genetics ; Laryngeal Neoplasms ; immunology ; prevention & control ; Newcastle disease virus ; immunology ; Plasmids ; Transfection ; Vaccines, DNA ; biosynthesis

OBJECTIVETo separate the cell subpopulation with high tumorigenic ability and study the biological characteristics of this subpopulation in laryngeal carcinoma cells.

METHODSHuman laryngeal carcinoma cells were obtained by primary tissue culture technique. CD44 and CD133 molecules were used as markers to isolate the CD44(+), CD133(+), CD44(+)CD133(+) and CD44(+)CD133(-) cell subpopulations from the laryngeal carcinoma cells by flow cytometry. A nude mouse tumor xenograft model was developed for the study of the tumorigenic effects of the different cell populations. 1 x 10(6), 1 x 10(5), 1 x 10(4) and 1 x 10(3) cells were injected into the left flank of the mice, respectively. The mice were observed for palpable tumor formation and were sacrificed at 4 weeks later to assess the tumor formation rate, tumor volume and tumor weight. Boyden chamber migration assay was used to determine the migration ability and immunochemistry was used to detect the expression of stem cell antigen SCA-1 and beta1-integrin. Semi-quantities RT-PCR and Western blot analysis were performed to detect the expression level of Bmi-1 in the different cell subpopulations.

RESULTSThe growth of subcutaneous tumors in nude mice showed that a tumor can be generated with 1 x 10(3) CD44(+)CD133(+) cells. When the same dose of 1 x 10(6) CD44(+)CD133(+) cells was injected into the mice, both the average weight and volume of the tumors were significantly higher than those generated from other cell subpopulations. Boyden chamber migration assay showed that the invasion ability of CD44(+)CD133(+) cells was significantly higher than that of other cell subsets. The results of immunochemical analysis showed an abundant expression of stem cell antigen SCA-1 and beta1-integrin in the CD44(+)CD133(+) cells. Semi-quantitative RT-PCR and Western blot analysis provided strong evidence that the Bmi-1 expression in CD44(+)CD133(+) and CD133(+) cells was very significantly higher than that in CD44(+), CD44(+)CD133(-) and control cells (P < 0.01).

CONCLUSIONOur findings demonstrate that CD44(+)CD133(+) subset cells in laryngeal carcinoma posses some biological characteristics of tumor stem cells, which may be the original cells of laryngeal carcinoma and may become a new target of tumor therapy.

AC133 Antigen ; Animals ; Antigens, CD ; analysis ; Antigens, Ly ; metabolism ; Cell Adhesion ; Gene Expression Regulation, Neoplastic ; Glycoproteins ; analysis ; Humans ; Hyaluronan Receptors ; analysis ; Integrin beta1 ; metabolism ; Laryngeal Neoplasms ; immunology ; metabolism ; pathology ; Male ; Membrane Proteins ; metabolism ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neoplasm Transplantation ; Nuclear Proteins ; genetics ; metabolism ; Peptides ; analysis ; Polycomb Repressive Complex 1 ; Proto-Oncogene Proteins ; genetics ; metabolism ; RNA ; metabolism ; Repressor Proteins ; genetics ; metabolism ; Tumor Burden ; Tumor Cells, Cultured

BACKGROUNDMounting evidence suggests that tumors are histologically heterogeneous and are maintained by a small population of tumor cells termed cancer stem cells. CD133 has been identified as a candidate marker of cancer stem cells in laryngeal carcinoma. This study aimed to analyze the chemoresistance of CD133(+) cancer stem cells.

METHODSThe response of Hep-2 cells to different chemotherapeutic agents was investigated and the expression of CD133 was studied. Fluorescence-activated cell sorting analysis was used to identify CD133, and the CD133(+) subset of cells was separated and analyzed in colony formation assays, cell invasion assays, chemotherapy resistance studies, and analyzed for the expression of the drug resistance gene ABCG2.

RESULTSAbout 1% - 2% of Hep-2 cells were CD133(+) cells, and the CD133(+) proportion was enriched by chemotherapy. CD133(+) cancer stem cells exhibited higher potential for clonogenicity and invasion, and were more resistant to chemotherapy. This resistance was correlated with higher expression of ABCG2.

CONCLUSIONSThis study suggested that CD133(+) cancer stem cells are more resistant to chemotherapy. The expression of ABCG2 could be partially responsible for this. Targeting this small population of CD133(+) cancer stem cells could be a strategy to develop more effective treatments for laryngeal carcinoma.

AC133 Antigen ; ATP Binding Cassette Transporter, Sub-Family G, Member 2 ; ATP-Binding Cassette Transporters ; genetics ; metabolism ; Antigens, CD ; genetics ; metabolism ; Antineoplastic Agents ; pharmacology ; Blotting, Western ; Carcinoma ; genetics ; metabolism ; Cell Line, Tumor ; Cisplatin ; pharmacology ; Flow Cytometry ; Fluorouracil ; pharmacology ; Glycoproteins ; genetics ; metabolism ; Humans ; Laryngeal Neoplasms ; genetics ; metabolism ; Neoplasm Proteins ; genetics ; metabolism ; Neoplastic Stem Cells ; cytology ; drug effects ; metabolism ; Paclitaxel ; pharmacology ; Peptides ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction

AIM To observe the changes of serum ROS,MDA,SOD levels in chronic renal failure (CRF) patients with damp-heat syndrome and to explore the intervention effect of Qingshen Granules (Hedyotis diffusae Herba,Salviae miltiorrhizae Radix et Rhizoma,Artemisiae scopariae Herba,etc.) on oxidative stress.METHODS Seventy cases of CRF patients with damp-heat syndrome were randomly and equally divided into control group and treatment group.Sixty cases completed the study,thirty-one cases in the treatment group,twenty-nine cases in the control group.Twenty other cases served as the normal group.The treatment group and the control group were given the basic treatment of western medicine and retention enema of TCM.The treatment group was added Qingshen Granules for eight weeks.The levels of U-Pro/24 h,Scr,BUN,estimated glomerular filtration rate (eGFR),serum ROS,MDA and SOD were measured before and after the treatment,and were compared with the normal group.RESULTS Both total effective rates on disease and on traditional Chinese medicine syndrome in the treatment group were 83.87％,significantly higher than those in the control group (51.72％ and 48.28％),respectively.The levels of U-Pro/24 h,Scr and BUN were obviously lower in the treatment group after the treatment,and eGFR was obviously higher as compared with those in the control group.Before the treatment,the levels of ROS and MDA in the treatment and control groups were significantly higher than those in the normal group,and the level of SOD was significantly lower;the levels of ROS and MDA were reduced in the treatment group after the treatment,and the level of SOD was increased;these changes in the treatment group were more significant than those in the control group.CONCLUSION Qingshen Granules can ameliorate clinical symptoms in CRF patients with damp-heat syndrome,decrease the levels of U-Pro/24 h,Scr and BUN,increase eGFR level,inhibit oxidative stress,and enhance antioxidant capacity,ultimately delay the development of renal fibrosis.

OBJECTIVETo investigate the age variations of head-face morphological traits of Hakka in Guangdong Province.

METHODSThirty-eight physical indices of head and face of 671 Hakka adults (151 urban males, 181 urban females, 162 rural males and 177 rural females) from Meizhou city of Guangdong were investigated. Twelve physical indices of head and face were calculated and counted its indices distributions. The age changes of morphological traits of head and face were analyzed.

RESULTSWith the age growing, the rate of eyefold of the upper eyelid was increased, and the Mongoloid fold decreased, the direction of eyeslits tended to level, nasal root height dropped, the zygomatic projection was more projected, the eye color turned to lighter, the upper red lips became thinner; medium-sized facial height, nasal height, upper lip skin height, physiognomic ear length, physiognomic ear breadth and breadth of alae nasi were decreased. The index of mouth breadth, morphological facial skin fold were positively correlated with the age. The index of minimum frontal breadth, face breadth, bigonial diameter, interocular breadth, external biocular breadth, lip height, thickness of lips and horizontal head circumference were negatively correlated with the age. With the age growing, morphological facial index and vertical cephalo-facial index was ascending. Moreover, length-height index of head, transverse frontoparietal index, zygomatico-frontal index and lip index were declining.

CONCLUSIONWith the age growing, the head and face measurements, physical indices and the proportion of observed indices have changed in Hakka in Guangdong province.

Adult ; Age Factors ; Aged ; Asian Continental Ancestry Group ; Cephalometry ; China ; Face ; anatomy & histology ; Female ; Head ; anatomy & histology ; Humans ; Male ; Middle Aged ; Sampling Studies ; Young Adult

OBJECTIVETo compare the characteristics of interbody fusion achieved using hat type cervical intervertebral fusion cage (HCIFC) with those of an autologous tricortical iliac crest graft, Harms cage and Carbon cage in a goat cervical spine model.

METHODSThirty-two goats underwent C(3, 4) discectomy and fusion in which the following were used: Group 1, autologous tricortical iliac crest bone graft (8 goats); Group 2, Harms cage filled with autologous iliac crest graft (8 goats); Group 3, Carbon cage filled with autologous iliac bone (8 goats); Group 4, HCIFC filled with autologous iliac graft (8 goats). Radiography was performed pre- and postoperatively and after 1, 2, 4, 8, and 12 weeks. At the same time points, disc space height, intervertebral angle, and lordosis angle were measured. After 12 weeks, the goats were killed and fusion sites were harvested. Biomechanical testing was performed in flexion, extension, axial rotation, and lateral bending to determine the stiffness and range of motion. All cervical fusion specimens underwent histomorphological analysis.

RESULTSOne week after operation, the DSH, IVA and LA of HCIFC and Carbon cage were statistically greater than those of autologous iliac bone graft and Harms cage. Significantly higher values for disc space height, intervertebral angle and lordosis angle were shown in cage-treated goats than in those that received bone graft over a 12-week period. The stiffness of Harms cage in axial rotation and later bending were statistically greater than that of other groups. Radiographic and histomorphologic evaluation showed better fusion results in cage groups than in autologous bone group.

CONCLUSIONSHCIFC can provide a good intervertebral distractability and enough biomechanical stability for cervical fusion.

Animals ; Biomechanical Phenomena ; Bone Transplantation ; methods ; Cervical Vertebrae ; diagnostic imaging ; physiopathology ; surgery ; Goats ; Ilium ; transplantation ; Internal Fixators ; Male ; Materials Testing ; Radiography ; Random Allocation ; Spinal Fusion ; instrumentation ; methods ; Transplantation, Autologous

Objective To explore the value of hippocampus hydrogen proton magnetic resonance spectrum analyzes (1 H-MRS) for the diagnosis of temporal lobe epilepsy (TLE) in children.Methods Singa HDe GE company to attain the 1.5T magnetic resonance imaging apparatus was used to analyze the conditions of 30 cases of children with TLE and the other 30 healthy controls received brain MRI and 1H-MRS check.The hippocampal N acetyl aspartate (NAA),creatine (Cr),and the choline (Cho) concentration were observed; NAA/(Cho + Cr),the ratio of NAA/Cr and NAA/Cho were analyzed.The analysis of biochemical components in the hippocampus was performed on the affected sides and the healthy sides of TLE group and the sides of the healthy control group,respectively.Results Hippocampus were anormal by combining MRI and MRS in 28 cases.Levels of NAA/(Cho + Cr),NAA/Cho,and NAA/Cr in the trouble sides of the TLE group had the remarkable statistical significance compared with the normal sides,and the healthy control group (all P ＜ 0.05),but there was no remarkable statistic significance between the normal sides and the healthy control group (all P ＞ 0.05).Nineteen cases had been confirmed by surgery,and postoperative pathological changes:brain surface were wavy,cortex were uneven;there were giant neurons,immature neurons,and scattered across the deformity of neurons in the cortex.A small amount of ectopic neurons can be seen in white matter.Conclusions In the TLE children,the reduction of NAA/(Cho + Cr) value of the side hippocampus may help diagnose the hipppcampal sclerosis earlier,and can provide significant evidence for the diagnosis and treatment of TLE in children.

The multiple systemic symptoms appearing during the convalescence of novel coronavirus infection(COVID-19)are called long-term symptoms after COVID-19,also named as long COVID.Traditional Chinese medicine(TCM)has unique advantages in the treatment of long COVID.In the view of Professor ZHANG Zhong-De,the novel coronavirus infection belongs to the category of damp-toxin pestilence in TCM.During the convalescence of novel coronavirus infection,the damp-toxin pathogen causes the dysfunction of qi,blood,yin and yang of zang-fu organs,and then results in the consumption of yang qi or the depletion of fluid and blood.Damp-turbidity obstructing qi movement and entering collaterals and then developing into blood stasis is the key pathogenesis of long COVID.Therefore,the therapeutic principle of harmonizing yin,yang,qi and blood of the five zang-organs is proposed,and the methods for transporting and transforming damp-toxin and phlegm-turbidity,regulating qi,activating blood and removing stasis should be adopted.According to the exuberance or decline of qi,blood and body fluid,and yin and yang of the five zang-organs,the Fuzheng Series Formulas(series formulas for supporting healthy qi)were recommended for the specific syndrome types:Fuzheng Shengbai Formula can be used for the syndrome of deficiency of original qi;Fuzheng Yifei Formula can be used for lung-spleen-kidney deficiency syndrome;Fuzheng Anshen Formula can be used for the syndrome of liver qi stagnation and heart-spleen deficiency.During the treatment,Professor ZHANG Zhong-De makes the modification of drugs indicated for the retention sites of damp-toxin and turbid qi,and addresses the importance of spleen and stomach in the whole treatment process by protecting spleen and stomach,so as to promote the rehabilitation of patients after novel coronavirus infection and improve their quality of life.

Objective To study the effect of silencing survivin on the growth of Hep-2 human laryngeal cancer cells in vitro and in vivo. Methods Hep-2 cells were transfected with pGCsilencer-siRNA-survivin(psi-survivin)by Lipofectamine 2000. The mRNA and protein expressions of survivin were detected by semi-quantitative RT-PCR and Western blot, respectively. Cell proliferation activity was measured by MTT assay. Apoptosis was assessed by flow cytometry. The implanted tumors were formed from injected Hep-2 cells in nude mice. After the tumor formation, psi-survivin was injected into peritumor tissues. The growth of tumor were observed. The tumor volume was calculated and the tumor growth curve was plotted. The expression of survivin in tumor tissues was detected by Western blot. The tumor cell apoptosis was observed by Tunel stainning. Results The sequence-specific siRNA of survivin inhibited the expressions of survivin mRNA and protein. The inhibition rates of survivin mRNA and protein expression were 54.4% and 37.0%respectively. Also the growth of Hep-2 cells was inhibited significanly,with a decrease by 71.7%. By the day 32 of tumor growth, the mean tumor volumes were ( 1443.9 ± 230.5 ) mm3 ( (-x) ± s ) in saline control group, ( 1348.5 ± 198.4) mm3 in plasmid-negative control group, and (624. 6 ± 188.4) mm3 in psi-survivin group,respectively (t = -5.917 ,P ＜0.01 ). In the implanted tumors injected with psi-survivin, survivin protein expression was down-regulated significantly,with a inhibition rate of 41.8%. Tunel stainning showed the apoptosis occurred in the implanted tunors. Conclusion Silencing survivin could significantly inhibit the growth of Hep-2 human laryngeal cancer cells in vitro and in vivo.

Objective:To observe serum osteoprotegerin (OPG),receptor activator of NF-kB ligand (RANKL) levels and the therapeutic effect of Rong Huang granules of patients with non-dialysis chronic kidney disease mineral and bone metabolism disorder (CKD-MBD) and kidney deficiency damp heat syndrome.Methods:70 cases of non-dialysis CKD-MBD with kidney deficiency damp heat syndrome,were randomly divided into treatment group and control group,the actual completion of 61 cases,30 cases in treatment group,31 cases in the control group;and a healthy normal group of 20 cases of patients was established.Two groups of patients were given symptomatic treatment,in addition,Rong Huang granules was plused in treating at treatment group and it was used three times a day,each time blunt one bag.The course of treatment was 8 weeks.The changes of kidney deficiency damp heat syndrome,blood urea nitrogen (BUN),serum creatinine (Scr),estimated glomerular filtration rate (eGFR),serum of calcium (Ca),phosphorus (P), alkaline phosphatase (ALP),parathyroid hormone (iPTH),OPG and RANKL levels were observed in two groups of patients.Results:The total effective rate in treatment group was significantly better than the control group(P<0.01).The integral value of syndrome decreased more significantly with the course of treatment increased of two groups of patients(P<0.01).Compared with the same period of therapy,the descender in the treatment group was significantly better than that in the control group(P<0.01).The levels of BUN, Scr,eGFR,Ca,P,iPTH and ALP were improved in the treatment group after treatment(P<0.05 or P<0.01),BUN and iPTH were improved in the control group(P<0.05 or P<0.01),other indexes were not improved(P>0.05).After treatment,BUN,Scr,eGFR,Ca, P,iPTH,ALP of treatment group were significantly better than the control group(P<0.05 or P<0.01).Compared with the normal group,the levels of OPG and RANKL were significantly higher in CKD-MBD patients (P<0.01).After treatment,serum OPG level, serum RANKL level and OPG/RANKL ratio were significantly improved than before treatment in treatment group(P<0.05 or P<0.01),and in the control group,only the OPG/RANKL ratio increased(P<0.01).After treatment,OPG,RANKL and OPG/RANKL ratio in the treatment group were obviously improved compared with them in the control group(P<0.05 or P<0.01).Conclusion:The levels of OPG and RANKL in non-dialysis CKD-MBD patients with kidney deficiency damp heat syndrome were higher than those in healthy people,and the ratio of OPG/RANKL was lower than that in healthy people.Rong Huang granules can ameliorate clinical symptoms,prevent calcium and phosphorus metabolism,improve renal function,the mechanism may be related to the ameliorate of serum OPG and the decrease of serum RANKL level and the raise of ratio of OPG/RANKL.