OBJECTIVE To search a method for identifying Clostridium perfringens and genotyping their toxin for gene diagnosis by multiplex PCR.METHODS The mutiplex PCR was developed with three sets of primers(designed) based on the sequences of three C.perfringens toxin genes(CP?,CP? and CPE) published in GenBank to identify C.perfringens and genotype their three toxin genes.RESULTS Three expected(sequences) were (obtained) successfully by multiplex PCR and identified by electrophoresis.CONCLUSIONS The(specific) sequences of C.perfringens could be amplified and their three genes of toxins could be identified by this multiplex PCR(system).Such method should be helpful for developing gene diagnosis well.

AIM:To test the hypothesis that wild-type p53 regulates the expression of p-glycoprotein. METHODS: While Hep3B cells lack expression of both p53 and retinoblastoma tumor suppressor genes because of deletions, were transfected to a wild-type (wt) p53 cDNA and control vector by a liposome method. After G418 selection, stable wt-p53 transformants, and control vector transformants (pNeo) were obtained. The expression of the p53,p21 and P-gp was analyzed by Northern or Western blot methods. In both transformants, cytotoxic effect of doxorubicin and mitomycin was evaluated by MTT assay, and accumulation of doxorubicin was detected by flow cytometer. RESULTS: In wt-p53 transformants, induction of transcriptionally active p53 was confirmed by the increase of P21 waf1/cip1 protein. Levels of P-gp reduced in the cells expressing wild-type p53 were linked to wt-p53 activity. The wt-p53 transfectants were more sensitive to doxorubicin and mitomycin compared with the pNeo transformants, and the accumulation of doxorubicin in the wt-p53 transfectants was 13 times as much as pNeo transformants. After both transformants were treated with doxorubicin at 0.2 mg/L for 24 hours, apparent apoptosis occurred in the wt-p53 transrormants. CONCLUSION: Restoration of wt-p53 activity in Hep3B leads to more sensitive to chemotherapeutic agents because of decrease of p-glycoprotein expression.

OBJECTIVETo explore clinical efficacy of double Endobutto reconstituting coracoclavicular ligament combined with repairing acromioclavicular ligament in stage I in treating acromioclavicular dislocation with Rockwood type III - V .

METHODSFrom January 2010 to September 2013, 56 patients with Rockwood type III - V acromioclavicular dislocation were treated by operation, including 20 males and 36 femlaes, aged from 32 to 52 years old with an average of 38.5 years old. Twenty-five patients were on the left side and 31 cases on the right side. The time from injury to operation was from 3 to 14 days, averaged 7 days. All patients were diagnosed as acromioclavicular dislocation with Rockwood type III - V, and double Endobutto were used to reconstituting coracoclavicular ligament, line metal anchors were applied for repairing acromioclavicular ligament. Postoperative complications were observed, Karlsson and Constant-Murley evaluation standard were used to evaluate clinical effects.

RESULTSAll patients were followed up from 8 to 24 months with average of 11 months. According to Karlsson evaluation standard at 6 months after operation,42 cases were grade A, 13 were grade B and 1 was grade C. Constant-Murley score were improved from (42.80±5.43) before operation to (91.75±4.27) at 6 months after operation. All items at 6 months after operation were better than that of preoperative items. Forty-eight patients got excellent results, 7 were moderate and only 1 with bad result. No shoulder joint adhesion, screw loosening or breakage were occurred during following up.

CONCLUSIONDouble Endobutto reconstituting coracoclavicular ligament combined with repairing acromioclavicular ligament in stage I for the treatment of acromioclavicular dislocation with Rockwood type III - V could obtain early staisfied clinical effects, and benefit for early recovery of shoulder joint function.

Acromioclavicular Joint ; injuries ; surgery ; Adult ; Bone Plates ; Bone Screws ; Female ; Humans ; Ligaments, Articular ; injuries ; surgery ; Male ; Middle Aged ; Reconstructive Surgical Procedures ; Shoulder Dislocation ; surgery ; Treatment Outcome

Objective To expore CT diagnostic values for hyaline-vascular type of giant lymph node hyperplasia(GLNH).Methods CT findings in 4 patients with hyaline-vascular type of GLNH confirmed by surgery and pathology were respectively analyzed and relevant literatures were reviewed.CT examination included plain and enhanced scan with contrast medium bolus injection.Results Lesions were located 2 at the posterior mediastinum,1 at the restroperitonum and 1 at the neck.The characteristic CT appearances were a large soft tissue mass round or oval in shape with homogenous density,well and regular margin.The masses were all markedly enhanced after contrast,their attenuation were similar to but slightly lower than the larger arteries in the same section.Conclusion GLNH is an uncommon disease and it′s clinical diagnosis is difficult.But CT scanning could showed some features.When CT found a mass with benign characters locating in lymph node regions,especially marked enhancement,Hyaline-vascular type of GLNH should be considered.

Objective To investigate the clinical features of cerebral infarction complicated with diabetes mellitus (DCI) compared withthose without diabetes mellitus (NCI). Methods 80 DCI and 86 NCI hospitalized patients were reviewed with their serumal glucose, lipid,uric acid, hemorheology and carotid ultrasound. Results The total cholesterol, low density lipoprotein and uric acid were significantly higherin DCI group than in NCI group (P<0.05), while high density lipoprotein was significantly lower (P<0.05). There were significant differencesbetween these two groups in Hemorheological indicators except hematokrit (P<0.01). The carotid intima media thickness (IMT) wassignificantly higher in the DCI group than in the NCI group (P<0.05). Conclusion DCI patients suffered in more serious lipid, uric acid,hemorheological disorder, and IMT compared with NCI.

Objective To establish a model of cervical spondylosis in rats. Methods Sixty SD rats (four months old) were ran-domly divided into control, muscle imbalance and posterior column instability group, ten rats in each group. Then the degeneration of X-ray film and motion function were evaluated by oblique board test. Results After analyzed the cervical films of control groups, the nature curve still existed, vertebral clearance had no abnormal. No ostensis spurs or ossifies in Luschka joint and joint processes were found. But in mus-cle imbalance and posterior column instability groups, the nature curve was disappeared or stiff, vertebral clearance were stenosis, osteosis spur formed, ossify or sclerosis emerged in Lnschka joint and joint process. Compared with control group in oblique board test, the muscle force decreased in muscle imbalance and posterior column instability group. There were no statistic significant difference between 2 and 4 months of control group, and also no difference in muscle imbalance and posterior column instability group, but there were significant differ-ence in 2 and 4 months of muscle imbalance and posterior column group. Conclusion Not only cervical posterior column instability but also muscle imbalance could result in cervical spendylosis, both muscle force balance and posterior column were important factors in maintaining spine stability.

Objective This paper is intended for establishing quality standard of Ershiwuwei Shanhu pill. Methods The TLC was used to indentify Pyrethrum tatsienense, Sesami nigrum semen, Aquilariae lignum resinatum, Caryophmlli flos, Acori calami rhizoma, Chebulae fructus, Aucklandiae radix and Glycyrrhizae radix et rhizome. For hydroxysafflor yellow A, the HPLC system consisted of WondaSil-C18 (250 mm×4.6 mm, 5μm), methanol-acetonitrile- 0.7% phosphoric acid (26:2:72) as mobile phase, flow rate of 1.0 ml/min, detection wavelength at 403 nm. For liquiritin, the HPLC system consisted of WondaSil-C18 (250 mm×4.6 mm, 5μm), acetonitrile (A)-0.05% phosphoric acid (B) as mobile phase, flow rate of 1.0 ml/min, detection wavelength at 237 nm. And for crocin-I, the HPLC system consisted of WondaSil-C18 (250 mm×4.6 mm, 5μm), methanol-water (49:51) as mobile phase, flow rate of 1.0 ml/min, detection wavelength at 440 nm. Results The methods of TLC were simple with strong specificity and good reproducibility. The results of HPLC showed that calibration curve was linear in the ranges of 0.046 7-0.233 8μg for hydroxysafflor yellow A, and 0.510 6-1.531 8μg for liquiritin and 0.048 1-0.340 5μg for crocin-I. The average recovery rate was 102.01%, 99.50%and 99.32%, respectively. Conclusion The new method is more appropriate for the quality control of Ershiwuwei Shanhu pill.

Objective To report the clinical outcome and prognostic factors for locally recurrent nasopharyngeal carcinoma(NPC)treated with intensity modulated radiotherapy(IMRT).Methods From January 2001 to August 2004,the data of 132 such NPC patients were analyzed retrospectively;104 male and 28 female with a median of 44.5 years(range 21-73 years).Ninety-eight patients(74.2%)were confirmed by biopsy as having NPC:9 with WHO TypeⅡand 89 WHO TypeⅢ.The other 34 patients were only diagnosed by MRI scan because of the extension/invasion was in the base of skull and/or cavernous sinus.Median interval time were 24 months(range 6-184 months).According to the 1992 Chinese Fuzhou Staging System:stageⅠ3.8 %,Ⅱ10.6 %,Ⅲ22.0% andⅣa 63.6%;T1 5.3%,T2 10.6%,T3 22.7% and T4 55.3%.Twenty-two patients had recurrence in the neck lymph nodes.IMRT was given with the sequential tomotherapy system(NOMOS Peacock systems)of 6 MV X-rays.Prescription dose was 60-70 Gy in GTV,with the fractional dose of 1.94-2.8 Gy.Sixty patients were also supplemented with two to six courses of cisplatin-based chemotherapy.Results The median volume of GTV was 39.5 cm~3(range 0.8-158.9 cm~3).The D95,V95,mean dose and fractionation dose of GTV was 66.9 Gy,98.3%,69.8 Gy and 2.32 Gy,respectively.The median follow-up time was 12 months(range,2-47 months).The 1-,2-and 3-year local progression-free rate was 96.4%,88.4% and 85.3%,respectively.The overall 1-,2-and 3-year survival rate was 6.5.9%,49.6% and 41.6%,respectively.Eleven patients developed distant metastases.Forty-seven patients were observed to devdop mucosa necrosis and/or massive hemorrhage in the nasopharynx.On univariate and multivariate analysis,fractional dose and vohane of GTV were significant prognostic factors for overall survival(P=0.016,0.009).Conclusions The local control and survival rate can be improved for patients with locally recurrent nasopharygeal carcinoma after treatment of intensity modulated radiotherapy.The fractional dose and volume of GTV are independent prognostic factors for the overall survival. The main death reasons are mucosa necrosis and/or massive hemorrhage in the nasopharynx.

Background Deficiency of neurotrophic factor is associated with the damage of optic nerve in glaucoma.Reaserches showed that ectopically applied neurotrophic factor has a transient neuroprotective effect in glaucoma model,and the viral expression of adeno-associated neurotrophie factor may provide long-term supplementation of neurotrophic factor and neuroprotection in tissues.Objective The present study Was to investigate the neuroproteetive effect of adeno-associated viral(AAV)-mediated brain-derived neurotrophic factor (BDNF)on retinal ganglion cells(RGCs)in DBA/2J mice with experimental glaucoma. Methods 10 clean DBA/2J mice were administered intravitreal injection with 1 microliter of AAV-BDNF-GFP in the left eyes at the age of 6 months,and the right eyes were injected with the same volume of saline solution as control.Intraocular pressure (IOP)was measured with Tonolab in the mice every month.Retinas were obtained after 3 months for the investigation of GFP expression in RGCs using fluorescence microscopy.Immunohistochemistry Was performed by applying TUJ1 and Cy3 antibodies to identify surviving RGCs. Results The IOP of DBA/2J mice were 11.90 mmHg and 11.40 mmHg in the right eyes and left eyes,respectively,at 4 months.The IOP of DBA/2J mice began to rise at 5 months and reached its peak in 8 month-old mice.There was no statistically significant difference in IOPs between the right eyes and the left eyes from 4 month-through 9 month-old mice(t=-1.78-0.61,P=0.11-0.90).Three months after intrlavitreal injection of AAV-BDNF-GFP,GFP was positively expressed in RGCs of retinas with the expression rate of 46.33%±8.08%.The over-expression of BDNF led to more RGCs survival than the control eyes (3168.13±1319.33/mm2 vs 2024.81±796.38/mm2,t=2.75,P=0.02). Conclusion These data suggest that BDNF can exert a protective effect in DBA/2J glaucoma mice.

Background Doxycycline is a broad spectrum antibiotic,and it is frequently used in the treatment of ocular surface diseases.Objective The purpose of the present study was to investigate the effect of doxycycline on the inhibition of cell proliferation in bovine corneal myofibroblasts in vitro and assess its contribution to ocular surface repairing mechanism.Methods Six fresh bovine corneas were collected.The corneal stromal layer was isolated by two-step method of 1.0 g/L and 2.0 g/L collegenase-1.Isolated cells were plated at mantaryay culture flask in 10％ FBS of RPMI-1640.Vimentin and alpha-smooth muscle actin (α-SMA) organization were evaluated by immunocytochemistry,and the cells with influoresccence staining for vimentin and α-SMA were identified as the corneal myofibroblasts.Doxycycline at the concentrations of 10,20,40,60,80 mg/L was added to the medium,respectively,in different concentrations of doxycycline groups.Dexamethasone (120 mg/L)was used in the same way in the positive control group,and no drug was used in the negative control group.Cell proliferation was evaluated by MTT and the cell cycle was analyzed by BD FACScan flow cytometer assay 24 hours and 48 hours after addition of any drug.Results The cells grew well and showed the positive response for vimentin and α-SMA.MTT assay showed that the A570values of bovine corneal myofibroblasts were gradually declined with the increase of the concentration of doxycycline and lapse of active time,showing statistically significant difference (Fconcentration =1233.778,P＜0.001 ; Ftime =227.564,P ＜ 0.001).And the difference between the two factors was also statistically significant (Ftime*concentration =51.656,P＜0.001).Flow cytometry cell cycle analysis showed that 24 hours after 10,20,40,60,80 mg/L doxycycline treated,the perentage of of corneal myofibroblast cell in G0-G1 phase was 82.85％,84.36％,85.18％,87.12 ％ and 89.31％,showing significant increase in comparison with 63.89％ of the negative control group (all P＜0.05),and that of 40 mg/L doxycycline group was near the positive control group.Forty-eight hours after 10,20,40,60,80 mg/L doxycycline treated,the perentage of of corneal myofibroblast cell in G0-G1 phase was 82.78％,86.15％,88.23％,89.57％,93.00％,with significant increase in comparison with 70.17％ of the negative control group (all P ＜ 0.01),and that of 40 mg/L doxycycline group was near the positive control group.Conclusions The growth of the bovine corneal myofibroblasts is inhibited by doxycycline in time-and dosedependent manner in the range from 10 mg/L to 80 mg/L,and 40 mg/L of doxycycline has an obviously inhibitory action as 120 mg/L dexamethasone.