Objective:To develop an EGFR-targeting nanobody engineered exosome drug delivery system and evaluate its antitumor efficacy for colorectal cancer.Methods:The HEK293T cell line stably expressing the pan-cancer inhibitor miR-204-5p, previously established by our group, was selected as a tool cell line to prepare miR-204-5p-enriched exosomes. Using metabolic glycoengineering combined with bioorthogonal reaction strategy, these exosomes were modified with the EGFR-specific nanobody 7D12. Western blot, electron microscopy, and dynamic light scattering were used to characterize the engineered exosomes. The tumor target potential of engineered exosomes was evaluated using immunofluorescence and RT-qPCR. The in vitro anti-tumor activities of engineered exosomes were evaluated using cell growth curves, colony formation, Transwell, and apoptosis analyses. The in vivo anti-tumor activity and safety of engineered exosomes were evaluated using a nude mouse xenograft tumor model. Results:The particle size of 7D12-hExo was (116.8±36.8) nm, with a potential of around -10 mV, and there was no significant change compared with the unmodified hExo. Immunofluorescence assay showed that the fluorescence intensity of the hExo group, 7D12-hExo group, and 7D12+7D12-hExo group were 48.4±3.9, 141.0±6.6, and 38.7±3.2 in EGFR + HCT116 cells, respectively. Compared with the hExo group, the fluorescence intensity of HCT116 cells in the 7D12-hExo group was significantly enhanced ( P＜0.05). Compared with the 7D12-hExo group, the fluorescence intensity in HCT116 cells in the 7D12+7D12-hExo group was significantly decreased ( P＜0.05). However, there was no significant difference in the uptake of hExo and 7D12-hExo in EGFR - SW620 colorectal cancer cells. The number of cell clones, invasion, and migration of HCT116 cells in the hExo (204) group was 215.0±14.0, 862.3±61.4, and 1 197.0 ± 36.7, respectively, with an apoptosis rate of (14.1±1.4)%. The number of cell clones, invasion, and migration of HCT116 cells in the 7D12-hExo (204) group was (65.0±15.1), (232.0±27.9), (725.7±32.7), respectively, with an apoptosis rate of (29.3±1.0)%. The 7D12-hExo (204) significantly inhibited the proliferation, invasion, and migration ability of HCT116 cells ( P＜0.05), resulting in promoting the apoptosis of HCT116 cells ( P＜0.05). Nude mouse experiments showed that 7D12-hExo (204) significantly inhibited the growth of tumors transplanted with HCT116 cells, with the inhibition rate being 82.8%. However, there was no significant change in mouse weight, and H&E staining of major organs such as heart, liver, spleen, lung, and kidney did not show any abnormalities. Conclusion:Naturally miR-204-5p-loaded exosomes were successfully modified with nanobody 7D12, which can efficiently deliver miR-204-5p into EGFR + tumor cells, thereby exerting good anti-tumor therapeutic effects.

Objective:To develop an EGFR-targeting nanobody engineered exosome drug delivery system and evaluate its antitumor efficacy for colorectal cancer.Methods:The HEK293T cell line stably expressing the pan-cancer inhibitor miR-204-5p, previously established by our group, was selected as a tool cell line to prepare miR-204-5p-enriched exosomes. Using metabolic glycoengineering combined with bioorthogonal reaction strategy, these exosomes were modified with the EGFR-specific nanobody 7D12. Western blot, electron microscopy, and dynamic light scattering were used to characterize the engineered exosomes. The tumor target potential of engineered exosomes was evaluated using immunofluorescence and RT-qPCR. The in vitro anti-tumor activities of engineered exosomes were evaluated using cell growth curves, colony formation, Transwell, and apoptosis analyses. The in vivo anti-tumor activity and safety of engineered exosomes were evaluated using a nude mouse xenograft tumor model. Results:The particle size of 7D12-hExo was (116.8±36.8) nm, with a potential of around -10 mV, and there was no significant change compared with the unmodified hExo. Immunofluorescence assay showed that the fluorescence intensity of the hExo group, 7D12-hExo group, and 7D12+7D12-hExo group were 48.4±3.9, 141.0±6.6, and 38.7±3.2 in EGFR + HCT116 cells, respectively. Compared with the hExo group, the fluorescence intensity of HCT116 cells in the 7D12-hExo group was significantly enhanced ( P＜0.05). Compared with the 7D12-hExo group, the fluorescence intensity in HCT116 cells in the 7D12+7D12-hExo group was significantly decreased ( P＜0.05). However, there was no significant difference in the uptake of hExo and 7D12-hExo in EGFR - SW620 colorectal cancer cells. The number of cell clones, invasion, and migration of HCT116 cells in the hExo (204) group was 215.0±14.0, 862.3±61.4, and 1 197.0 ± 36.7, respectively, with an apoptosis rate of (14.1±1.4)%. The number of cell clones, invasion, and migration of HCT116 cells in the 7D12-hExo (204) group was (65.0±15.1), (232.0±27.9), (725.7±32.7), respectively, with an apoptosis rate of (29.3±1.0)%. The 7D12-hExo (204) significantly inhibited the proliferation, invasion, and migration ability of HCT116 cells ( P＜0.05), resulting in promoting the apoptosis of HCT116 cells ( P＜0.05). Nude mouse experiments showed that 7D12-hExo (204) significantly inhibited the growth of tumors transplanted with HCT116 cells, with the inhibition rate being 82.8%. However, there was no significant change in mouse weight, and H&E staining of major organs such as heart, liver, spleen, lung, and kidney did not show any abnormalities. Conclusion:Naturally miR-204-5p-loaded exosomes were successfully modified with nanobody 7D12, which can efficiently deliver miR-204-5p into EGFR + tumor cells, thereby exerting good anti-tumor therapeutic effects.

Objective:To evaluate the bone hook pulling technique in reduction of valgus-impacted femoral neck fractures.Methods:A retrospective study was conducted to analyze the clinical data of 12 patients who had been treated from September 2021 to September 2022 for valgus-impacted femoral neck fractures at Department of Orthopaedics and Traumatology, Honghui Hospital. There were 6 males and 6 females with an age of (43.8±10.1) years. By the Garden classification, the 12 cases were all type Ⅰ; by the Pauwels classification, 10 cases were type Ⅰ and 2 cases type Ⅱ. All patients were definitively diagnosed preoperatively by radiographs and CT images. The bone hook pulling technique was used to extend the bone hook from the lateral side to the medial side of the lesser trochanter, pull the bone hook to the lateral side, and reset the fracture with the help of pulling force and the action of the surrounding soft tissue hinge. The fractures were then fixed with the Femoral Neck System (FNS). Length of surgical incision, number of fluoroscopy for bone hook-related operations, operation time, quality of fracture reduction, fracture healing time, functional recovery of the hip and shortening of the femoral neck at the last follow-up were recorded.Results:The 12 valgus-impacted femoral neck fractures were successfully reset by the hook pulling technique. In this cohort, the length of surgical incision was (4.6±0.7) cm, the number of fluoroscopy for bone hook-related operations (4.3±0.7) times, and the operation time (54.3±4.2) min. The 12 patients were followed up for (11.5±4.2) months postoperatively and the fracture healing time (4.2±0.7) months. According to the Garden score, the quality of postoperative fracture reduction was assessed as grade Ⅰ in 10 cases and as grade Ⅱ in 2 cases. According to the Harris hip score, the hip function was assessed as excellent in 10 cases and as good in 2 cases at the last follow-up. The length of femoral neck shortening was (1.17±0.68) mm at the last follow-up in the 12 patients; no complications related to fracture reduction were observed.Conclusion:In the reduction of valgus-impacted femoral neck fractures, bone hook pulling technique shows advantages of operational simplicity, a high rate of successful reduction, and satisfactory clinical effects.

Objective:To evaluate the bone hook pulling technique in reduction of valgus-impacted femoral neck fractures.Methods:A retrospective study was conducted to analyze the clinical data of 12 patients who had been treated from September 2021 to September 2022 for valgus-impacted femoral neck fractures at Department of Orthopaedics and Traumatology, Honghui Hospital. There were 6 males and 6 females with an age of (43.8±10.1) years. By the Garden classification, the 12 cases were all type Ⅰ; by the Pauwels classification, 10 cases were type Ⅰ and 2 cases type Ⅱ. All patients were definitively diagnosed preoperatively by radiographs and CT images. The bone hook pulling technique was used to extend the bone hook from the lateral side to the medial side of the lesser trochanter, pull the bone hook to the lateral side, and reset the fracture with the help of pulling force and the action of the surrounding soft tissue hinge. The fractures were then fixed with the Femoral Neck System (FNS). Length of surgical incision, number of fluoroscopy for bone hook-related operations, operation time, quality of fracture reduction, fracture healing time, functional recovery of the hip and shortening of the femoral neck at the last follow-up were recorded.Results:The 12 valgus-impacted femoral neck fractures were successfully reset by the hook pulling technique. In this cohort, the length of surgical incision was (4.6±0.7) cm, the number of fluoroscopy for bone hook-related operations (4.3±0.7) times, and the operation time (54.3±4.2) min. The 12 patients were followed up for (11.5±4.2) months postoperatively and the fracture healing time (4.2±0.7) months. According to the Garden score, the quality of postoperative fracture reduction was assessed as grade Ⅰ in 10 cases and as grade Ⅱ in 2 cases. According to the Harris hip score, the hip function was assessed as excellent in 10 cases and as good in 2 cases at the last follow-up. The length of femoral neck shortening was (1.17±0.68) mm at the last follow-up in the 12 patients; no complications related to fracture reduction were observed.Conclusion:In the reduction of valgus-impacted femoral neck fractures, bone hook pulling technique shows advantages of operational simplicity, a high rate of successful reduction, and satisfactory clinical effects.

Objective To investigate the clinical efficacy of endoscopic submucosal dissection (ESD)for the treatment of colorectal tumors, and to analyze risk factors affecting operation time. Methods A retrospective study was conducted using data of 74 cases with colorectal tumor,who underwent ESD in Department of Gastroenterology of Wuhan University Renmin Hospital from January 2014 to September 2015. The clinical efficacy of ESD, occurrence of complications and follow-up results were summarized,and the risk factors of operation time were analyzed. Results The rate of ESD en bloc resection and histological complete resection was 97.30%(72/74)and 89.19%(66/74), respectively. Among the 8 cases of histological non-curative resection, 2 cases received appended surgical procedures because of deep invasion of tumor(SM2),6 cases were given close follow-up according to the pathological result of adenomas. Among the 74 cases, no acute hemorrhage or pneumoperitoneum occurred. Four cases (5.41%, 4/74)had postoperative delayed bleeding, and were successfully treated by endoscopic hemostasis. Four cases(5.41%, 4/74)had intraoperative perforation, and were successfully treated by endoscope. No recurrence or abnormal lesions occurred during the follow-up of 15-35 months. Multivariate linear regression analysis showed that tumor size was a main risk factor for ESD operation time(P=0.000). Conclusion ESD, as a minimally invasive treatment, is safe and effective for the treatment of colorectal tumors,and the tumor size is a main risk factor of ESD operation time.

Objective This study was carried out to investigate the effect of myocardial insulin resistance on expression of p38 mitogen-activated protein kinase (MAPK) in ischemic heart failure in rat.Methods Male sprague-dawley rats were subjected either to ligation of the left anterior descending artery (LAD) (n =24) or to sham operation (n =24).After two weeks,cardiac size and function were determined by echocardiography.Glucose and fatty acid (FAO) oxidation rates as well as insulin response were measured in the isolated working heart.The protein expression of p38MAPK was evaluated by Western blotting.Results The infarcted hearts were dilated and had a reduced ejection fraction (ejection fraction ＜0.50).The basal glucose oxidation was preserved,but the fatty acid oxidation was significantly reduced.Insulin effect on substrate oxidation was significantly impaired for both the decrease in fatty acid oxidation and the increase in glucose oxidation.The protein expression of p38MAPK in infracted hearts wasfisigni cantly reduced(P＜0.05).Condusion Myocardial infarction in rats caused partial insulin resistance at the level of substrate oxidation,which was associated with cardiac contractile dysfunction and the expression of p38MAPK.