OBJECTIVETo establish a drug-resistance cell line of human glioma mediated by MGMT.

METHODSSimulated the clinical usage of BCNU to establish a BCNU-resistant human glioma subline by cyclic exposing the U251 parent cells to a constant concentration of BCNU. The resistance index and the expression of MGMT mRNA of U251/BCNU were detected and compared the difference of in vitro proliferation between U251 and U251/BCNU.

RESULTSA subline--U251/BCNU was successfully established in about 4-month culture, which had a stable resistance to BCNU. U251/BCNU cells showed 17-fold higher resistance to BCNU than did U251 cells by MTT assay, while U251/BCNU cells expressed MGMT mRNA. The doubling time of U251 and U251/BCNU had no statistical difference.

CONCLUSIONA drug-resistance cell line of human glioma mediated by MGMT is established, which could provide experimental basis for further studies on the resistance mechanism and reversal methods of glioma chemotherapy.

ATP-Binding Cassette, Sub-Family B, Member 1 ; biosynthesis ; Brain Neoplasms ; pathology ; Cell Line, Tumor ; DNA Modification Methylases ; biosynthesis ; genetics ; Drug Resistance, Neoplasm ; genetics ; Glioma ; pathology ; Humans ; O(6)-Methylguanine-DNA Methyltransferase ; metabolism ; physiology

Objective To evaluate the development of the sphincter muscle complex(SMC)and defecation function in pediatric patients with congenital anorectal malformations(ARM).Methods A total of 64 children underwent MRI,among whom 39 were patients with ARM,and the others were patients without ARM undergoing MRI because of other dieases.The dimensions of the SMC in different planes were evaluated with different sequences and coils.The relationship between the SMC development and the defecation function was investigated.Results In control group,the absolute value of SMC width was (3.63?0.22)mm,which had a high correlation with age(r=0.998,P0.05).The SMCs in intermediate ARM patients[muscle index(MI)=0.47?0.05]and low ARM patients(MI=0.49? 0.05)were well developed.The SMCs in a portion of patients with high ARM(MI=0.28?0.06)were poorly developed,when MI≤0.18,anorectal contraction pressurewas significantly lower(t=3.55, P0.18[(0.85?0.20)vs(2.24?1.02)kPa].The length of anal canal with high-pressure[(10.88?3.64)vs(20.26?4.34)mm]was shorter(t=5.18,P0.18,the anorectal angle was less than 90 degrees,and normal continent function was found in 21 of 23 cases(91%).Conclusion MRI can be employed to evaluate the development of SMC in patients with ARM,MI was an objective criteria to evaluate the development of SMC.When MI≤0.18, maldevelopment of SMC will be highly suspected.

OBJECTIVETo investigate the effectiveness of phosphorothioate multidrug resistance gene 1 (MDR1) antisense oligodeoxynucleotides (MDR1-AS) suppressing MDR1 expression in multidrug-resistant glioma cell line C6/adr.

METHODSThe glioma cell line C6/adr served as the tested model in vitro, MDR1-AS (5'-CTCCATCACCACCTC-3'), complementary to the -9- +6 sequence of first exon, was synthesized and phosphorothioate-modified. As control of sequence specificity, MDR1-S (5'-GAGGTGGTGA TGGAG-3') was used. Both antisense and sense oligodeoxynucleotides were transduced to C6/adr cells by lipofectin. The cytotoxity of MDR1-AS was tested using morphological observation and 3- (4,5-dimethylthiazol-2-yl) -2, 5-diphenyl tetrazolium bromide assay. Semi-quantitative reverse transcription polymerase chain reaction was used to monitor the expression levels of the MDR1 mRNA in the different groups. The positive rate of the MDR1 gene product P glycoprotein (P-gp) was determined by flow cytometry assessment.

RESULTSNo cytotoxicity of MDR1-AS was observed. The MDR1 mRNA expression level was decreased from 106% to about 30.44% 48 h after MDR1-AS treatment. The P-gp positive rate of MDR1-AS treated C6/adr cells decreased from 100% to 32.77%, with that of C6/adr cells considered as 100%.

CONCLUSIONSMDR1-AS can effectively inhibit MDR1 expression in the C6/adr cell line at both the mRNA and protein level, and may be an alternative treatment of drug-resistant gliomas.

ATP-Binding Cassette, Sub-Family B, Member 1 ; biosynthesis ; genetics ; Cell Line, Tumor ; Drug Resistance, Multiple ; Drug Resistance, Neoplasm ; Glioma ; genetics ; metabolism ; Humans ; Oligodeoxyribonucleotides, Antisense ; pharmacology ; RNA, Messenger ; biosynthesis ; genetics

Objective: To summarize the clinical application patterns in acupuncture-moxibustion treatment of AD by reviewing the clinical literatures on acupuncture-moxibustion for Alzheimer disease (AD) published between January 2009 and December 2019. Methods: China National Knowledge Infrastructure (CNKI), Wanfang Academic Journal Full-text Database (Wanfang), Chongqing VIP Database (CQVIP), Chinese Medicine Acupuncture-moxibustion Information Database, PubMed Medical Data Retrieval Service System, Springer Database and Ovid Technologies (OVID) were retrieved to screen clinical studies of acupuncture-moxibustion treatment of AD according to the inclusion and exclusion criteria to conduct quantitative, clustering and association analyses. Results: In acupuncture-moxibustion treatment of AD, the frequently used points were Baihui (GV 20), Zusanli (ST 36), Sishencong (EX-HN 1), Taixi (KI 3), Sanyinjiao (SP 6), and Neiguan (PC 6) in the descending order. Regarding meridians, the most frequently used one was the Governor Vessel, followed by the Stomach Meridian of Foot Yangming and Gallbladder Meridian of Foot Shaoyang. From the perspective of body regions, the points in the head-face region and the lower-limb region had the highest frequencies, followed by the upper-limb, back and chest-abdomen regions. The point group, Baihui (GV 20) and Sishencong (EX-HN 1)-Neiguan (PC 6)-Sanyinjiao (SP 6), showed the most significant association, and the group winning the second place was Baihui (GV 20) and Sishencong (EX-HN 1)-Neiguan (PC 6)- Zusanli (ST 36). The clustering analysis showed that the commonly used point pairs included Zusanli (ST 36)-Sishencong (EX-HN 1) and Taixi (KI 3)-Sanyinjiao (SP 6), which were closely associated with Baihui (GV 20). By analyzing the three commonly used acupuncture-moxibustion methods, acupuncture plus medication was found achieving the best result in the total effective rate and mini-mental state examination (MMSE) score, followed by monotherapy of electroacupuncture therapy, and these two methods were superior to acupuncture alone (P<0.05); the scores of MMSE, Alzheimer disease assessment scale-cognitive section (ADAS-cog) and activity of daily living scale (ADL) showed significant improvements after treatment (all P<0.01). Conclusion: In the acupuncture-moxibustion prescriptions for AD, the main points are Baihui (GV 20), Sishencong (EX-HN 1), Neiguan (PC 6), Zusanli (ST 36), Sanyinjiao (SP 6) and Taixi (KI 3). Monotherapy of acupuncture has the highest frequency amongst the treatment methods, but its effective rate is lower than that of acupuncture plus medication and monotherapy of electroacupuncture.

The aim of this study was to evaluate the effect of all-trans retinoic acid (ATRA) on cell adhesion molecule expression and adhesion capacity of bone marrow stromal cells (BMSC) in patients after conditioning treatment for peripheral blood stem cell transplantation (PBSCT). BMSC of 27 patients before and after conditioning treatment for PBSCT were cultured in vitro. After treated with ATRA at 0.01, 0.1, or 1 micromol/L, expression of intercellular adhesion molecule-1 (ICAM-1) protein and vascular adhesion molecule-1 (VCAM-1) protein were detected by flow cytometry, and soluble ICAM-1 (sICAM-1) protein was determined by using radioimmunoassay. Then BMSC was co-cultured with CD34+ cells, and adhesion rate of BMSC to CD34+ cells was measured. The results showed that after pretreatment with conditioning regimen for PBSCT, the expressions of ICAM-1 and VCAM-1 proteins in BMSC and the expression level of sICAM-1 protein in supernatant of BMSC culture were down-regulated, and the adhesion rate of BMSC to CD34+ cells was decreased, after administration of ATRA, the expression of ICAM-1 protein in BMSC, sICAM-1 protein in culture medium and adhesion rate of BMSC to CD34+ cells all increased significantly, but expression of VCAM-1 protein changed no significantly. It is concluded that the ATRA can partly restore adhesion function of BMSC injured by pretreatment for PBSCT and contribute to hematopoietic reconstitution.
Adolescent ; Adult ; Antigens, CD34 ; Antineoplastic Agents ; pharmacology ; Bone Marrow Cells ; metabolism ; pathology ; Cell Adhesion ; drug effects ; Cell Adhesion Molecules ; biosynthesis ; genetics ; Child ; Coculture Techniques ; Hematologic Neoplasms ; metabolism ; pathology ; therapy ; Humans ; Middle Aged ; Peripheral Blood Stem Cell Transplantation ; Stromal Cells ; metabolism ; pathology ; Tretinoin ; pharmacology ; Tumor Cells, Cultured ; Vascular Cell Adhesion Molecule-1 ; biosynthesis ; genetics

The aim of this study was to evaluate the effect of dimethyl amiloride (DMA), a specific inhibitor of Na(+)/H(+) exchanger-1 (NHE-1), on intracellular pH value (pHi), proliferation and apoptosis of HL-60/ADM cells in vitro. After treatment with DMA at different doses, pHi of HL-60 and HL-60/ADM cell lines were determined by using pH-sensitive fluorescence dye BECEF-AM; the rate of growth inhibition of cells was detected with MTT assay; cell cycle was detected by flow cytometric DNA analysis; cell apoptosis was observed with terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). The results showed that pHi in HL-60/ADM cells was higher than that in HL-60 cells. After treatment with DMA at different doses, pHi decreased, the rate of growth inhibition and the rate of apoptotic cells in HL-60/ADM cells were all higher than those in HL-60 cells. Meanwhile, after treatment with DMA during 100 micromol/L to 150 micromol/L, the increase amplitude of G(0)/G(1) phase cells and the decrease amplitude of S + G(2)/M cells in HL-60/ADM cells were higher than those in HL-60 cells. It is concluded that by causing intracellular acidification, the NHE-1-specific inhibitor DMA inhibits proliferation of HL-60/ADM cells and induces apoptosis of HL-60/ADM cells, and the degree of this growth inhibition of HL-60/ADM cells is higher than that of HL-60 cells.
Amiloride ; analogs & derivatives ; pharmacology ; Apoptosis ; drug effects ; Cell Cycle ; drug effects ; Cell Proliferation ; drug effects ; Doxorubicin ; pharmacology ; Drug Resistance, Neoplasm ; HL-60 Cells ; Humans ; Hydrogen-Ion Concentration ; Sodium-Hydrogen Exchangers ; antagonists & inhibitors

In order to study the influence of different gelatin concentrations, and lymphocyte isolation liquid on primary culture of umbilical cord blood-derived adhesive cells (hCBACs), the red blood cells of umbilical cord blood was separated by 3% and 6 % gelatin for detecting the effectiveness of sedimentation, then the adhesion rate at 48 hours, the day of initial expansion and the rate of culture success were detected for hCBACs cultured with CD34(+) cells after the mononuclear cells were separated by 6% gelatin followed by Ficoll and Percoll, and the morphological characteristics and growth status were observed by invert microscopy. Cytochemistry stain for nonspecific esterase stain (NSE), peroxidase (POX), periodic acid Schiff reaction (PAS) and alkali phosphatase (ALP) and immunocytochemistry labeling for CD31, CD45, CD68 and fibronectin (Fn) were detected. The results showed that 6 % gelatin was better than that 3% gelatin for red blood sedimentation. The Percoll was predominant over Ficoll in adhesion rate at 48 hours, the day of initial expansion, the time of initial formation of adhesive cell colony units, the time of maximal numbers of adhesive cell colony units, the the cell fusion time and ratio of culture success. 60% fibroblast-liked cells, 36% macrophage liked cells and 4% small-round cells were observed in cells isolated by both isolated methods. The cytochemistry stain for NSE, POX, PAS and ALP was similar in two groups, the difference was not statistically significant between these two groups. The immunocytochemistry labeling for CD31, CD45, CD68 and Fn was also similar in both groups and the difference was also not statistically significant between these two groups. It is concluded that the combination of 6% gelatin with Percoll is an ideal separation method for primary culture of hCBACs, which provides basic information for clinical application.
Cell Separation ; methods ; Cells, Cultured ; Fetal Blood ; cytology ; Gelatin ; administration & dosage ; pharmacology ; Humans ; Lymphocytes ; cytology

OBJECTIVETo observe the effects of inhibiting stromal cell derived factor-1 (SDF-1) expression by RNA interference (RNAi) on adhesion and drug sensitivity of Jurkat cells co-cultured with bone marrow stromal cells.

METHODSSDF-1 specific short hairpin RNA (shRNA) expressing plasmid was transferred into cultured human acute leukemic bone marrow stromal cells, positive clones were isolated by screening G418 resistance (Group A) , SDF-1 protein level in culture supernatant was detected by enzyme-linked immunosorbent assay (ELISA). The adhesion rates to bone marrow stromal cells layer and the drug sensitivity to doxorubicin of co-cultured Jurkat cells were detected by cell counting and MTT assay, respectively. The un-transfected bone marrow stromal cells of acute leukemia patient (Group B) or normal subject (Group C) were taken as control.

RESULTSThe level of secreted SDF-1 protein (pg/10(5) cells/week) in the supernatants of Group A, B and C were 1920 +/- 205, 12,370 +/- 1355 and 6620 +/- 770, respectively. Of co-cultured Jurkat cells in Group A, B and C, the adhesion rates after 24 h co-culturing were (28.8 +/- 2.6)%, (57.4 +/- 3.8)% and (45.2 +/- 4.0)%, respectively, and the IC50 values of doxorubicin were 585, 6162 and 1758 nmol/L, respectively.

CONCLUSIONDown-regulating SDF-1 expression of bone marrow stromal cells by RNAi reduces adhesion rates and enhances drug sensitivity to doxorubicin of their co-cultured Jurkat cells.

Bone Marrow Cells ; metabolism ; Cell Adhesion ; Cells, Cultured ; Chemokine CXCL12 ; genetics ; metabolism ; Coculture Techniques ; Drug Resistance, Neoplasm ; Gene Expression ; Humans ; Jurkat Cells ; RNA Interference ; Stromal Cells ; metabolism

OBJECTIVETo study the effects of RNA interference inhibiting stromal cell derived factor-1 (SDF-1) expression on the proliferation and apoptosis of co-cultured Jurkat cells.

METHODInhibition of SDF-1 expression by RNA interference (RNAi) was achieved by transferring SDF-1 specific short hairpin RNA (shRNA) expressing plasmid into cultured human acute leukemic bone marrow stromal cells. Resistant clones were obtained by G418 selection (group A). The concentration of SDF-1 protein in culture supernatant was detected by enzyme-linked immunosorbent assay (ELISA). The population double time (PDT), cell cycles, apoptosis rates and the expressions of PCNA, Bcl-2/Bax, Fas/FasL of co-cultured Jurkat cells were detected by cells counting, flow cytometry. TdT-mediated dUTP nick-end labelling (TUNEL) and immunocytochemistry (ICC), respectively. The un-transfected acute leukemic (group B) and normal (group C) bone marrow stromal cells were taken as controls.

RESULTSThe content of SDF-1 protein in supernatant of group A\[(384 +/- 41) pg/ml] was significantly lower than that in group B[(2474 +/- 271) pg/ml] or group C[(1324 +/- 154) pg/ml]. As group A compared with group B and group C, the PDT of co-cultured Jurkat cells was prolonged (group A: 42 h, vs group B: 29 h, group C: 33 h), and G(0)/G(1) stage cells increased [group A: (28.47 +/- 2.39)%, vs group B: (19.43 +/- 2.80)%, group C: (27.15 +/- 2.07)%], S stage cells decreased [group A: (25.57 +/- 1.90)%, vs group B: (74.48 +/- 3.23)%, group C: (60.99 +/- 2.33)%], G(2)/M stage cells increased [group A: (45.96 +/- 3.24)%, vs group B: (6.09 +/- 1.96)%, group C: (11.86 +/- 1.98)%], the apoptosis rate increased [group A: (15.2 +/- 0.8)%, vs group B: (5.4 +/- 0.7)%, group C: (9.5 +/- 0.4)%], and the expressions of PCNA, Bcl-2, Fas decreased; whereas the expressions of Bax and FasL were increased.

CONCLUSIONThe inhibition of SDF-1 expression in bone marrow stromal cells inhibits the proliferation and promotes the apoptosis of co-cultured Jurkat cells.

Apoptosis ; genetics ; Bone Marrow Cells ; metabolism ; Cell Proliferation ; Cells, Cultured ; Chemokine CXCL12 ; genetics ; Coculture Techniques ; Gene Expression ; Humans ; Jurkat Cells ; RNA Interference ; Stromal Cells ; metabolism ; Transfection

OBJECTIVETo improve clinicians' ability of diagnosing testicular torsion.

METHODSWe reviewed the data of a case of testicular torsion that resulted in necrosis because of delayed presentation and repeated misdiagnosis, and analyzed its anatomic features, clinical manifestations, ultrasound results, the causes of misdiagnosis and relevant literature.

RESULTSThe patient presented 5 hours after the onset of symptoms, complaining of severe paroxysmal pain in the lower left abdomen, accompanied with nausea and vomiting, and was twice misdiagnosed as having enterospasm or ureteral calculus at two different hospitals. Fifteen hours later, surgical exploration revealed an about 900-degree testicular torsion in the spermatic cord, which necessitated orchiectomy for non viability of the testis. Postoperative pathological examination confirmed testicular necrosis and diffused hemorrhage in the testis and epididymis.

CONCLUSIONTimely presentation, correct diagnosis and proper treatment are keys to saving the affected testis. Color Doppler ultrasound is an ideal option for the definite diagnosis of acute scrotal diseases, and it offers a valuable guidance for related surgery as well.

Adult ; Diagnostic Errors ; Humans ; Male ; Necrosis ; Spermatic Cord Torsion ; diagnosis ; Testis ; pathology