ObjectiveTo explore the effects of glycomacropeptide (GMP) in human milk and formula milk on proliferation ofbifidobacterium infantis and their dose-response relationship.Methods Casein was isolated from the milk of 30 healthy postpartum women from Guangzhou Women and Children's Medical Center in September 2014, and hydrolyzed by rennet to obtain GMP, which was then purified by ultrafiltration and ion exchange chromatography. Human milk GMP and cow milk GMP (0, 250, 500, 1 000, 1 500, 2 000 and 3 000 mg/L) were added tobifidobacterium infantis liquid medium, and cultured under anaerobic conditions. Concentration of bacteria was measured by turbidimetric microplate assay (detection of OD600 nmvalue of medium). Difference of proliferative activities ofbiifdobacterium infantis in human milk GMP and cow milk GMP was compared with independent samplest-test.ResultsPurified human milk GMP concentration was 1 712.20 mg/L, with a purity of 80.3%. Increasing the cow milk GMP initial concentration in the culture medium at 250-2 000 mg/L could increase the concentration and proliferation rate ofbiifdobacteria infantis. When cultured at 36 h with GMP of various concentrations, the proliferation ofbiifdobacteria infantis maintained at a logarithmic phase. Therefore, 36 h was chosen as the test time point to compare the proliferation ofbifidobacterium infantis. At 36 h, when GMP in the medium was 1 000, 1 500, 2 000 and 3 000 mg/L, concentrations ofbiifdobacteria infantis in human milk GMP were 2.255±0.036, 2.583±0.088, 2.877±0.080 and 3.219±0.081, respectively, which were significantly higher than those in cow milk GMP (2.115±0.053, 2.312±0.064, 2.542±0.090 and 2.894±0.076;t=4.867, 5.569, 6.192 and 6.516; allP<0.01).Conclusions Both human milk GMP and cow milk GMP can promote the proliferation ofbiifdobacterium infantisin vitro, and the proliferative activity in human milk is greater than in cow milk at the same concentration of GMP.

Objective To investigate the dynamic changes of endoplasmic reticulum stress-related molecules including glucose regulated protein (GRP78), C/EBP homologous protein (CHOP), and caspase-12 in sciatic nerve of diabetic rats and explore its mechanisms.Methods Rats were randomly divided into normal control group (NC) and diabetes mellitus group (DM) that were induced by intraperitoneal injection of Streptozocin after 4 weeks of high-fat chow feeding.Sciatic nerves were isolated for three times at 4 weeks, 8 weeks and 12 weeks after induction of diabetes.The expressions of GRP78, CHOP,and caspase-12 were detected with quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot analyses.The morphology of sciatic nerve was investigated with electron microscope.Results With the extension of the course, demyelinating and axonal injury appeared in sciatic nerve of diabetic rats.The expressions of GRP78 mRNA and protein in DM group were significantly higher than NC group at 4 weeks and 8 weeks after induction of diabetes(P ＜0.05, P ＜0.01).The expressions of CHOP mRNA and protein in DM group were significantly higher than NC group at 8 weeks and 12 weeks after induction of diabetes (P ＜ 0.05).The expressions of caspase-12 mRNA and protein in DM group were significantly higher than NC group at 8 weeks after induction of diabetes(P ＜ 0.05, P ＜ 0.01).Conclusions Endoplasmic reticulum stress-related molecules (GRP78, CHOP, and caspase-12) contributed to the peripheral nerve injury of diabetic rats, and displayed dynamic changes.

Objective To study the preparation of quercetin compound cream and establish its standard of quality control. Methods The composition of recipe and manufacturing technique were designed.The content of components were determined by HPLC,and its stability tests were carried out.Results The product was a kind of yellow smooth cream.The linear ranges were 0.053-1.696 μg for quercetin(r=0.999 9),0.053-1.696 μg for 8-methoxypsoralen(r=0.999 8)and 0.100-1.000 μg for beta-methasone(r=0.999 9).The average recovery rate were 99.83%,99.52%,and 99.74% of quercetin,8-methoxypsoralen,and beta-methasone(n=9).After 12 months′long term stability test,all the 3 batches of sample preparations were in line with relevant regu-lations.Conclusion The designed recipe was reasonable,and the manufacturing technique was feasible,with stable and controllable quality.

Objective To investigate the expression and clinical significance of intergrin linked kinase (ILK) in pancreatic carcinoma.Methods ILK protein was detected by immunohistochemistry and Western blotting in 60 cases of pancreatic carcinoma and 32 cases of normal pancreatic tissue,and the relationship with the clinicopathological characteristics were analyzed.Results Immunohistochemistry showed ILK was expressed in cytoplasm and membrane of pancreatic carcinoma cells and the positive rate was 65% (39/60),which was significantly higher than 18.75% (6/32) of normal pancreatic tissue(P ＜0.05 ).Western blotting showed the expression of ILK in pancreatic carcinoma tissue was 303933±195116,which was significantly higher than 144613±30074 of normal pancreatic tissue(P＜0.05 ).In pancreatic carcinoma,the expression of ILK was correlated with clinical stage and lymph metastasis(P＜0.05 ),but not correlated with tumor cell differentiation(P＞0.05 ).Conclusions ILK protein was highly expressed in pancreatic carcinoma tissue and it was correlated with the degree of malignancy.

Objective To investigate the application value of identification of the scalp surface locations of cerebral ischemia lesions before direct revascularization for moyamoya disease and to design surgical approaches according to this by using the fusion of single photon emission computed tomography ( SPECT) cerebral perfusion imaging with CT imaging. Methods The clinical data of 13 adult patients with ischemic-type moyamoya disease underwent superficial temporal artery-middle cerebral artery bypass surgery were analyzed retrospectively. SPECT cerebral perfusion imaging was fused with CT imaging of the same machine before procedure. The lesions of ischemia were located on the cortical surface. The surgical approaches were designed at the center of the ischemic lesions. The patients were followed up for 6 to 12 months after procedure. The improvement of clinical symptoms and cerebral perfusion of the patients were observed after operation. Results One patient had perioperative cerebral hyperperfusion syndrome,and the others did not have any perioperative complications. At one-month follow-up, the improvement of symptoms in 4 patients were excellent,in 5 were good,in 4 were fair,and none was poor. At 6 to 12 month follow-up,the improvement of symptoms in 9 patients were excellent,in 4 were good,and none was poor. The postoperative visual SPECT cerebral perfusion imaging analysis suggested that the cerebral perfusion was improved significantly as compared with before procedure in all patients. Quantitative analysis:There was significant difference in target ischemic lesions between preoperative Fb and postoperative Fb ([2. 13±1. 06]% vs. [4. 13±2. 09]%;P<0. 05). There was significant difference between preoperative Fb and Fe ([2. 46±1. 97]% vs. [2. 13±1. 06]%;P<0. 05). The postoperative BFCR was [67. 57±3. 78]%( >50%) , which indicated that the efficacy of the procedure was remarkable. The superficial temporal arteries fed to brain of the patients were observed after procedure by using the head CT angiography. The postoperative head MRI reexamination showed no new infarcts occurred at 6 months. Conclusion Combine SPECT cerebral perfusion imaging with CT imaging to design surgical approach for superficial temporal artery-middle cerebral artery bypass surgery may improve the efficacy and reduce the risks of operation.

Objective To investigate the value of imaging-pathology fusion guided targeted cryoablation for localized prostate cancer.Methods Between June 2014 and February 2017,64 patients undergoing targeted cryoablation for localized prostate cancer were retrospectively collected and analyzed.The average age was 77.8 years old,ranging 45-87 years old.The preoperative valne of PSA ranged from 5.1 to 19.8ng/ml,with mean of 15.3 ng/ml,including 6 case of cT1c,35 cases of cT2a,and 23 cases of cT2b,and 35 cases of Gleason score 6,8 cases of 3 + 4 and 21 cases of 4 + 3.All patients had good continence and 11 cases had good erectile function before operation.All the patients received MRI or contrast-enhanced ultrasonography examinations before biopsies,and the sites and range of cryoablation were determined by MRI or contrast-enhanced ultrasonography and biopsy pathological results.Results All the patients underwent the surgery successfully.The average postoperative hospital stay was 1.6 days,ranging 1 to 3 days,including 22 cases discharged within 24 h after surgery.The operative duration ranged from 85 to 152 min,with mean of 99.6 min.The satisfaction rate of the patients was 100％.Seven patients had Clavien Ⅰ or Ⅱ postoperative complications,and recovered after symptomatic treatment.The follow-up duration ranged from 0.5 to 32.2 months,with median of 11.8 months.All patients were alive without new metastasis.Eight patients developed biochemical recurrence,5 of those underwent intermittent endocrine therapy,resulting in stable PSA levels.Conclusion Imaging-pathology fusion guided targeted cryoablation was safe and effective for localized prostate cancer with the advantages of few complications,high satisfaction rate and quick recovery.

In this article, HPLC fingerprint analysis method for total flavonoids from pollen of Brassica campestris L.was established., The HPLC fingerprint was performed on Waters C18 column (250 mm × 4.6 mm, 5 μm), eluted gradiently with the mixture of acetonitrile and 0.4% phosphoric acid aqueous solution at a flow rate of 0.8 mL·min-1. The column temperature was 40℃. The detection wavelength was 320 nm. The HPLC standard fingerprint of total flavonoids from pollen of Brassica campestrisL. was established, and 16 common peaks were calibrated. The method was simple, stable, and reproducible. It could be applied for quality control of total flavonoids from pollen of Brassica campestris L.

Objective To investigate the effects of lipopolysaccharides (LPS) in different concentrations on the proliferation and interleukin(IL)-6,IL-1 β and tumor necrosis factor-α(TNF-o) secretion of intestinal epithelial cells (IEC-6) of rats in vitro.Methods IEC-6 of rats were divided into normal group (0 mg/L,group A),0.1 mg/L group (group B),0.5 mg/L group (group C),1.0 mg/L group (group D),5.0 mg/L group (group E) and 10.0 mg/L group(group F).Different groups cells were exposed to LPS with different concentrations for 3 h,5 h and 7 h.Thiazolyl blue(MTT) was performed to investigate the proliferation of IEC-6.The concentrations of IL-6,IL-1 β and TNF-α in culture supernatant were detected by enzyme linked immunosorbent assay(ELISA).Results The proliferation rate of IEC-6 was gradually lower while the concentration of LPS increased.After co-culture with LPS 3h and 5 h,the proliferation rates of group B,group C,group D,group E and group F had no significant difference with those of group A (all P ＞ 0.05);after co-culture with LPS 7 h,the proliferation rates of group B,group C,and group D had no significant difference with those of group A (all P ＞ 0.05);the proliferation rates of group E and group F had significant difference with those of group A(t =4.216,P =0.014;t =14.991,P =0.000).The proliferation rates of group E and group F were lower after co-culture with LPS 5 h than 7 h,and there were significant differences (t =2.576,P =0.033;t =2.975,P =0.018);but there was no significant differences between group E and group A after co-culture with LPS 5 h (P ＞ 0.05).Group B,group C,group D,group E and group F all had a significant higher level of IL-6 than group A after co-culture with LPS 3 h,5 h and 7 h(all P ＜0.01).In addition,group E had the highest level of IL-6 at all time points.And the peak level of IL-6 rose after co-culture with LPS 5 h.The TNF-α level and IL-1 β level of group B,group C,group D,group E and group F all had no significant differences than that of group A after co-culture with LPS 3 h,5 h and 7 h (all P ＞ 0.05).Conclusions In a certain concentration,incubation time range,the proliferation rates of IEC-6 cells were gradually lower while the concentration of LPS increased.Co-cultured IEC-6 cells with LPS(0-10.0 mg/L) can stimulate them secrete to IL-6.The highest level of IL-6 was of group E after 5 h co-culture.LPS had no effects on TNF-α and IL-1 β level of IEC-6 cells cultural supernatant.So 5.0 mg/L concentration of LPS stimulating IEC-6 cells for 5 h can be chosen to build the IEC-6 inflammatory models.

Objective To detect the effects of bifidobacterium or bifidobacterium cultured supernatant on the mRNA expression of tumor necrosis factor receptor-associated factor 6 (TRAF6),glycogen synthase kinase-3β (GSK-3β) and the miRNA-146a in rat small intestinal epithelial cell(IEC-6) induced by lipopolysaccharide (LPS).Methods IEC-6 in logarithmic phase were randomly divided into LPS group,cultured supernatant group and inactivated bacteria group.All the 3 groups were exposed to 5 mg/L LPS for 5 hours,and then 1 mL sterile saline was added in LPS group and culturing continued for 24 hours ; 1 mL bifidobacterium cultured supernatant was added in cultured supernatant group and culturing continued for 24 hours;1 mL inactivated bifidobacterium 1 x 1010 CFU/L added in inactivated bacteria group and continued culturing for 24 hours.The mRNA expressions of TRAF6,GSK-3 β and miRNA-146a were detected by reverse transcription-polymerase chain reaction (RT-PCR).Results The level of TRAF6,GSK-3 β of culture supematant group (0.72 ± 0.05,0.46 ± 0.14) were all lower than LPS group (1.01 ± 0.14,1.02 ± 0.25),but the level of miRNA-146a(3.05 ± 0.40) was higher than that in LPS group(1.01 ± 0.12),and there were significant differences between them (t =5.278,6.316,13.218,P =0.000).The level of GSK-3 β of inactivated bacteria group(0.59 ±0.13) was significantly lower than that in LPS group(t =4.837,P =0.000).The levels of TRAF6 and miRNA-146a of inactivated bacteria group(1.05 ±0.11,0.78 ±0.22) had no significant differences with LPS group (t =0.732,1.463,P ＞ 0.05).The level of TRAF6 of cultured supernatant group was lower than that in inactivated bacteria group,and the level of miRNA-146a was higher than that in inactivated bacteria group,and there were significant differences between 2 groups (t =6.009,14.687,P =0.000).Conclusions Bifidobacterium cultured supernatant and inactivated bacteria both have certain protective effect on the IEC-6 induced by LPS.One of the protective mechanisms of bifidobacterium cultured supernatant may be achieved by elevating the expression of miRNA-146a,and decreasing the levels of inflammation related factor TRAF6 and damage related factor GSK-3β.The protective effects of inactivated bifidobacterium may be achieved by decreasing the level of damage related factor GSK-3β.

Objective To establish a mouse model of genital human papillomavirus (HPV) pseudovirion (PsV) transmission and evaluate the protective potency of HPV16 VLP vaccine.Methods HPV16 PsV with the encapsidated luciferase expressing plasmid Luc were generated from 293FT cells and purified by size-exclusion chromatography.The endpoint titers of HPV16 PsV-Luc were determined on 293FT cells, denoted as TRLU/mL.For in vivo genital challenge, mice were synchronized in a diestrus-like status by a subcutaneous injection with 0.1 μg β-estradiol and then with 3mg DepoProvera after 24 hours.Six hours prior to HPV16 PsV-Luc challenge, deeply anesthetized mice were intravaginally pretreated with 50 μL of 4%nonoxynol-9 ( N-9 ).HPV16 PsV-Luc was thoroughly mixed with 20 μL solution containing 4%carboxymethylcellulose ( CMC ) and intravaginally instilled using a positive-displacement pipette.Forty-eight hours after PsV-Luc challenge, mice were anesthetized and D luciferin was intravaginally instilled.After 3 minites, bioluminescence was measured with a cryogenically cooled Xenogen IVIS camera system.Then,the murine genital challenge model was used to determine the potency that HPV16 VLP vaccine is efficient at preventing HPV infection.Results HPV16 PsV-Luc was generated and purified from 293FT cells.HPV16 PsV-Luc was verified to containe L1 and L2 protein by Western blot.HPV 16 PsV-Luc successfully infected vaginal epithelial cells of mouse and the murine genital challenge model was established.To achieve consistent bioluminescence, the minimal dose of HPV16 PsV-Luc was 1.7 ×104 TRLU.The protective potency of HPV16 VLP vaccine was shown using this murine model.Our data showed that immune serum with over neutralizing antibody titer of 256-fold was sufficient to confer protection against HPV PsV genital infection .Conclusion The murine genital challenge model of HPV16 was successfully established, and the model is used to evaluate the potency of HPV16 VLP vaccine against in vivo genital HPV16 PsV challenge.Our model will benefit for the investigation of HPV neutralization and the potency evaluation of the HPV vaccine .