Objective:To construct bacterial cytoplasmic membrane nanovesicles(BMV)with overexpressing programmed death 1(PD-1),denoted as BMV-PD-1 and evaluate the targeting efficacy of BMV-PD-1 towards transplanted lung tumor tissues in mice.Methods:The fusion plasmid ClyA-PD-1-EGFP fused by PD-1 and Cytolysin A(ClyA)was transferred into Escherichia coli BL21-Codonplus through plasmid transformation.Laser confocal microscopy,SDS-PAGE,and WB were used to detect the expression of the fusion protein ClyA-PD-1-EGFP.Bacterial membranes were extracted and processed with an extruder to generate BMV-PD-1.TEM and NTA were utilized to assess the morphology,size distribution,and zeta potential of BMV-PD-1,while WB was used to verify the presence of PD-1 protein.Laser confocal imaging was conducted to monitor the uptake of BMV-PD-1 by Lewis lung cancer cells.A C57BL/6J mouse subcutaneous transplant tumor model of LLC lung cancer cells was constructed,and the tumor targeting of BMV-PD-1 was evaluated by small animal imaging system.Results:Laser confocal microscopy images demonstrated that the plasmid ClyA-PD-1-EGFP was transferred into BL21-Codonplus and successfully expressed into protein.SDS-PAGE results suggested that ClyA-PD-1-EGFP was overexpressed in BL21-Codonplus.WB analysis indicated that PD-1 was expressed in bacteria and highly expressed in BMV-PD-1(P<0.001).NTA and TEM analyses revealed that BMV-PD-1 were spherical vesicles with a diameter of(145±14)nm and a negative surface charge.Laser confocal imaging showed that the high expression of PD-1 significantly increased the uptake of BMV-PD-1 by lung cancer cells(P<0.01).In vivo imaging of small animals further confirmed that the high expression of PD-1 can effectively improve cancer targeting of BMV-PD-1(P<0.01).Conclusion:In this study,bacterial plasma membrane nanovesicles BMV-PD-1 with high PD-1 expression are successfully constructed,and it is found that PD-1 overexpression markedly improve the mouse lung cancer xenograft tissue targeting specificity of BMV-PD-1,laying the groundwork for further development of BMV-PD-1 as a carrier for targeted drug delivery systems in tumors.

Objective To evaluate the role of μ-opioid receptor (MOR) in attenuation of bone cancer pain by anti-nerve growth factor (anti-NGF) in rats. Methods Part Ⅰ Sixty female SD rats weighing 200-220 g were randomly divided into 4 groups (n = 15 each): sham operation group (group S), sham operation + anti-NGF group (group SN), bone cancer pain group (group P) and bone cancer pain+ anti-NGF group (group PN) . Bonecancer was induced by intra-tibial inoculation of 1 × 105 Walker 256 breast cancer cells in group P and PN. Group S and SN received injection of PBS 10 μl. APE 10 catheter was inserted at L2,3 interspace into the epidural space 13 days after cancer cell inoculation. Three days after the catheter was successfully placed, group SN and PN received intrachecal (IT) injection of anti-NGF 10 μg (in normal saline (NS) 10 μl) and group S and P IT injection of NS 10 μl twice a day for 5 consecutive days. The number of spontaneous flinches (NSF), paw withdrawal latency (PWL) and paw withdrawal threshold (PWT) were measured before and 13, 16, 18, 21 day after cancer cell inoculation. The animals were sacrificed at 21 day after cancer cell inoculation and the spinal cord dorsal horn and dorsal root ganglion were removed for determination of MOR and MOR mRNA expression. Part Ⅱ Thirty female SD rats weighing 200-220 g were randomly divided into 2 groups (n = 15 each): bone cancer pain + anti-NGF group (group PN) and bone cancer pain + naloxone + anti-NGF group (group PNN). Bone cancer was induced by intratibial inoculation of 1 × 105 Walker 256 breast cancer cells. APE 10 catheter was inserted at L2-3 interspace into the epidural space 13 days after cancer cell inoculation. Three days after the catheter was successfully placed,group PN received IT injection of anti-NGF 10 μg (in NS 10 μl) and group PNN IT injection of naloxone 10μg (in NS 25 μl) and 0.5 h later IT injection of anti-NGF 10 μg (in NS 25μl) twice a day for 5 consecutive days. NSF,PWL and PWT were measured before and 13, 16, 18, 21 days after cancer cell inoculation. Results Part ⅠCompared with group S, no significant change was found in NSF, PWL and PWT in group SN, and in MOR and MOR mRNA expression in group SN and PN (P ＞ 0.05), NSF was significantly increased, PWL shortened, PWT decreased at 13-21 days after inoculation in group P and PN, and MOR and MOR mRNA expression was down-regulated in group P (P ＜ 0.05 or 0.01). Compared with group P, NSF was significantly decreased, PWL prolonged, PWT increased, MOR and MOR mRNA expression was up-regulated in group PN at 18-21 days after inoculation (P ＜ 0.05 or 0.01). Part Ⅱ Compared with group PN, NSF was significantly increased, PWL shortened, PWT decreased at 18-21 days after inoculation in group PNN (P ＜ 0.05 or 0. 01). Conclusion The mechanism by which anti-NGF attenuates bone cancer pain in rats is related to the activation of MOR.

Objective To detect the c fos proto oncogene expression in the hypothalamus of the mice which underwent water immersion and the effects of propofol Methods Twenty four mice were divided randomly into three groups:group A (normal control), group B (water immersion) and group C (water immersion+propofol) The group C was subdivided into two sub groups: in group C1 3mg/kg propofol was administered intraperitoneally 10min after water immersion; in group C2 3mg/kg propofol was given intraperitoneally 10min before water immersion The cerebral c fos positive cells were detected by S P immunohistochemical assay and the gastric ulcer index was calculated using Guth method Results Animal in group A had no gastric mucosa injury The gastric ulcer index increased significantly (6 2?2 1) with the obvious bleeding points in gastric mucosa and the rate of c fos gene positive cell increased markedly (64 2%?2 1%) in group B as compared with those in group A The gastric ulcer index and c fos gene expression rate decreased greatly in group C1 (3 2?1 0, 21 8%?3 2%) and group C2 (2 1?0 9, 18 4%?3 5%) respectively (P