Objective: To constructCSF1R+/-mice and to analyze their genotypes, so as to provide animal model basis for disease pathological mechanism and drug target. Methods : A linearized targeting vector was designed according to Cre/Loxp system. A Loxp site was inserted upstream of the 5th exon of theCSF1Rgene, and a neomycin resistance box with Loxp sites on both sides was inserted downstream of the 5th exon. The linearized targeting vector was electroporated into embryonic stem cells. The correctly targeted embryonic stem cells were injected into the blastocysts of C57BL/6J mice to obtain chimeric mice, which were bred with Zp3-Cre mice. The newborn mice were numbered 9-14 days after birth and their tails were cut. The DNA of the mice was extracted, and the genotype of the mice was identified by polymerase chain reaction and agarose gel electrophoresis. The expression of CSF1R in mouse macrophages was detected by flow cytometry. The expression of CSF1R in mouse tissues was detected by Western blot. Results: The results of agarose gel electrophoresis showed that 453 bp bands were amplified in wild type mice, and 453 bp and 650 bp bands were amplified in heterozygous mice. The results of flow cytometry showed that the expression of CSF1R in peritoneal macrophages and bone marrow-derived macrophages of CSF1R heterozygous mice was lower than that of WT group(P<0.05). The results of Western blot showed that the expression of CSF1R in spleen, kidney and brain tissue of CSF1R heterozygous group was lower than that of WT group(P<0.05). Conclusion CSF1R+/-mice are successfully constructed, reproduced and identified, which provides an animal model basis for further revealing the potential mechanism of CSF1R in immune regulation.

Objective : To construct a humanized mouse model of the interleukin-9 receptor(IL-9R) coding DNA sequence(CDS) gene and to verify the genotype and IL-9R expression in mice. Methods : The CRISPR/Cas9 genome editing technology was used to replace the exon 2-7 fragment of the il-9r gene in mouse embryonic stem cells with the corresponding human IL-9R sequence. After verifying the completion of the gene fragment replacement, tetraploid embryos were constructed and microinjected back into the oviducts of surrogate mice. Through surrogacy by female mice, homozygous humanized mice were obtained. DNA was extracted from the homozygous humanized mice IL-9R CDS gene, and their genotypes were identified by agarose gel electrophoresis after PCR amplification. Western blot was used to detect the expression of IL-9R in the spleen and thymus of homozygous humanized mice with either wild-type(WT) or IL-9R gene humanization. Results : Gel electrophoresis after PCR amplification showed that mice with only a 1 805 bp band amplified using WT primers were wild-type, while mice with 2 553 bp and 2 340 bp bands amplified using 5KI and 3KI primers, respectively, were homozygous humanized mice with IL-9R CDS gene. Western blot results indicated that the tissues of homozygous humanized mice model with IL-9R CDS gene expressed IL-9R significantly. Conclusion The humanized mouse model with IL-9R CDS gene has been successfully constructed and characterized.

Objective: To construct microRNA(miR)-22 gene knockout(miR-22-/-) mice using CRISPR/Cas 9 technology, to breed miR-22-/- mice and to identify their genotypes. Methods : In this experiment, CRISPR/Cas 9 technology was used to construct miR-22-/- genetically engineered mice. After gene identification, the F0 generation miR-22-/- mice were mated with wild-type mice in the same litter to obtain F1 generation miR-22-/- mice. The miR-22 knockout efficiency was analyzed at the RNA level by real-time fluorescence quantitative polymerase chain reaction(qPCR). Western blot was used to detect the interaction between miR-22 and target genes. Results : miR-22-/- mice were successfully constructed using CRISPR/Cas 9 technology, gene identification was performed on the bred mice, and three stable genotypes of miR-22+/+,miR-22+/-, and miR-22-/- were identified. The real-time fluorescence quantitative PCR detection results confirmed that miR-22-/- mice showed almost no expression of miR-22 in the heart, liver, lung, kidney, spleen, and thymus tissues compared to wild-type mice in the same litter. Western blot analysis showed that the relative expression level of NLRP3 protein in miR-22-/- mouse tissues was lower than that in wild-type mice. Conclusion A miR-22-/- mouse model is successfully constructed, and stable genetic homozygous miR-22-/- mice is obtained. This indicates that miR-22 has an inhibitory effect on the downstream target gene NLRP3.

Objective: To explore the breeding and genotyping of CCR2 knockout mice, and to verify the applicability of the polymerase chain reaction(PCR) method for genotype detection of CCR2 knockout mice. Methods: The introduced CCR2 pure male mice and wild-type female mice were mated and bred to produce the offspring generation, the obtained F1 generation heterozygous mice were continued to be mated. DNA was extracted by clipping the tail tissues of the mice at the age of 2 weeks, the target gene fragment was amplified by PCR, and the genotypic results were determined by agarose gel electrophoresis. The proportion of purebred progeny carrying the CCR2 knockout gene was increased by genetic crosses, the effect of CCR2 knockout in the progeny mice was verified by using Western blot against major immune cells and key organs, and flow cytometry was used to detect whether the knockout of the CCR2 gene had any effect on the function of the immune system by targeting the major immune cells. Results: CCR2 knockout mice were successfully bred and characterized, and three genotypes of F2 generation mice were obtained: CCR2+/+, CCR2+/-, and CCR2-/-. The offspring genotypes were identified by PCR, and Western blot showed extremely low CCR2 protein expression in CCR2 knockout mice. Flow analysis showed that CCR2 knockdown reduced the expression of CD4+T and Th1 cells in mouse spleen-derived T cells, but did not affect macrophage function. Conclusion Correct breeding and identification are important ways to get the pure CCR2 knockout mice, and PCR method for identifying mouse genotypes is simple, fast and reliable.

Objective:To explore the short-term clinical efficacy of arthroscopy-assisted surgical dislocation combined with autologous iliac bone graft for early and middle-stage femoral head necrosis in young and middle-aged patients.Methods:A retrospective study was conducted to analyze the clinical data of the 20 patients with early and middle-stage femoral head necrosis who had been treated by arthroscopy-assisted surgical dislocation combined with autologous iliac bone graft at Orthopaedic Medical Center, Haikou Hospital Affiliated to Xiangya School of Medicine, Central South University from January 2020 to December 2024. There were 12 males and 8 females, with an age of 31 (21, 46) years. The femoral head necrosis was due to hormones in 6 cases, to alcohol in 8 cases, to trauma in 3 cases, and to primary lesions in 3 cases. The International Bone Circulation Research Association staging: stage 2B in 2 cases, stage 2C in 5 cases, stage 3A in 7 cases, stage 3B in 3 cases, and stage 3C in 3 cases. The surgical time, intraoperative blood loss, and incidence of complications during follow-up were recorded in this cohort. The visual analogue scale (VAS) pain score, hip Harris score, Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) in this cohort were compared between pre-surgery, 3 months after surgery, and the last follow-up.Results:In this cohort, the surgical time was 134 (120, 160) minutes, the intraoperative blood loss 275 (250, 300) mL, and the follow-up duration (28.7±8.2) months. At pre-surgery, 3 months after surgery, and the last follow-up, respectively, the VAS pain scores were 6.0 (5.0, 7.0) points, 2.0 (1.0, 3.0) points, and 0.5 (0.0, 1.0) points; the WOMAC scores 45.5 (40.3, 46.8) points, 12.5 (10.5, 14.0) points, and 9.5 (8.3, 10.0) points; the Harris hip scores (58.1±4.8) points, (78.4±4.7) points, and (85.0±6.7) points. There were statistically significant differences in the pairwise comparisons between the 3 time points ( P<0.05). At the last follow-up, the therapeutic effects were evaluated according to the Harris hip score as excellent in 7 cases, as good in 10 cases, and as poor in 3 cases, giving an excellent and good rate of 85.0% (17/20). Two patients developed lower limb intermuscular vein thrombosis after surgery, 2 patients had delayed healing of greater trochanter osteotomy after nail removal, heterotopic ossification occurred in 2 patients, and further collapse of the femoral head occurred in 7 patients. No patient developed complications such as wound infection or nerve injury during the follow-up period. Conclusion:The arthroscopy-assisted surgical dislocation combined with autologous iliac bone graft can lead to good short-term clinical efficacy in the treatment of early and middle-stage femoral head necrosis in young and middle-aged patients, but complications such as femoral head collapse, delayed healing of greater trochanter osteotomy, heterotopic ossification, and lower limb intermuscular vein thrombosis still exist.

Objective:To explore the short-term clinical efficacy of arthroscopy-assisted surgical dislocation combined with autologous iliac bone graft for early and middle-stage femoral head necrosis in young and middle-aged patients.Methods:A retrospective study was conducted to analyze the clinical data of the 20 patients with early and middle-stage femoral head necrosis who had been treated by arthroscopy-assisted surgical dislocation combined with autologous iliac bone graft at Orthopaedic Medical Center, Haikou Hospital Affiliated to Xiangya School of Medicine, Central South University from January 2020 to December 2024. There were 12 males and 8 females, with an age of 31 (21, 46) years. The femoral head necrosis was due to hormones in 6 cases, to alcohol in 8 cases, to trauma in 3 cases, and to primary lesions in 3 cases. The International Bone Circulation Research Association staging: stage 2B in 2 cases, stage 2C in 5 cases, stage 3A in 7 cases, stage 3B in 3 cases, and stage 3C in 3 cases. The surgical time, intraoperative blood loss, and incidence of complications during follow-up were recorded in this cohort. The visual analogue scale (VAS) pain score, hip Harris score, Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) in this cohort were compared between pre-surgery, 3 months after surgery, and the last follow-up.Results:In this cohort, the surgical time was 134 (120, 160) minutes, the intraoperative blood loss 275 (250, 300) mL, and the follow-up duration (28.7±8.2) months. At pre-surgery, 3 months after surgery, and the last follow-up, respectively, the VAS pain scores were 6.0 (5.0, 7.0) points, 2.0 (1.0, 3.0) points, and 0.5 (0.0, 1.0) points; the WOMAC scores 45.5 (40.3, 46.8) points, 12.5 (10.5, 14.0) points, and 9.5 (8.3, 10.0) points; the Harris hip scores (58.1±4.8) points, (78.4±4.7) points, and (85.0±6.7) points. There were statistically significant differences in the pairwise comparisons between the 3 time points ( P<0.05). At the last follow-up, the therapeutic effects were evaluated according to the Harris hip score as excellent in 7 cases, as good in 10 cases, and as poor in 3 cases, giving an excellent and good rate of 85.0% (17/20). Two patients developed lower limb intermuscular vein thrombosis after surgery, 2 patients had delayed healing of greater trochanter osteotomy after nail removal, heterotopic ossification occurred in 2 patients, and further collapse of the femoral head occurred in 7 patients. No patient developed complications such as wound infection or nerve injury during the follow-up period. Conclusion:The arthroscopy-assisted surgical dislocation combined with autologous iliac bone graft can lead to good short-term clinical efficacy in the treatment of early and middle-stage femoral head necrosis in young and middle-aged patients, but complications such as femoral head collapse, delayed healing of greater trochanter osteotomy, heterotopic ossification, and lower limb intermuscular vein thrombosis still exist.

To sum up integrative nursing experience of a case with chronic refractory wound formation caused by drug extravasation.The essentials of integrative nursing are:structured nursing intervention of"assessment-management-treatment"based on the Triangle of Wound Assessment;determination of the timing for integrative nursing according to the theory of TCM sores and ulcers;implementation of copper board scraping method to promote circulation of qi and blood;use of Huo-long Comprehensive Moxibustion Therapy to promote muscle regeneration.With the help of the cooperation of the multidisciplinary specialist nursing team,the wound was completely healed after 59 days of integrated traditional Chinese and Western medicine nursing interventions.

Objective To investigate the expression of Acyl-CoA synthetase long-chain family member 4(ACSL4)in liver cancer and its role in regulating ferroptosis and proliferation of liver cancer cells.Methods Clinical samples of liver cancer and adjacent normal liver tissues were examined for malondialdehyde(MDA)contents and for expressions of mRNA and protein expressions of ACSL4 and proliferating cell nuclear antigen(PCNA)using RT-qPCR and Western blotting.Human liver cancer Huh-7 cells were treated with Erastin(a ferroptosis inducer),Fer-1(a ferroptosis inhibitor),or both,and the changes in expression levels of MDA,ACSL4 and PCNA were detected,and the cell proliferation was assessed with plate cloning assay.Results MDA contents were lower and ACSL4 and PCNA expressions were higher significantly in liver cancer tissues than in adjacent liver tissues.In Huh-7 cells,Erastin treatment significantly inhibited mRNA and protein expressions of ACSL4 and PCNA,suppressed cell proliferation,and increased MDA contents.Fer-1 alone did not produce significant effect on cell viability but reversed the effect of Erastin on ACSL4 and PCNA expressions,cell proliferation and MDA contents.Conclusion ACSL4 level is significantly overexpressed in liver cancer.Erastin increases MDA contents and down-regulates ACSL4 expression,thereby promoting ferroptosis and inhibiting proliferation of liver cancer cells,and these effects can be reversed by Fer-1.

Objective To investigate the expression of Acyl-CoA synthetase long-chain family member 4(ACSL4)in liver cancer and its role in regulating ferroptosis and proliferation of liver cancer cells.Methods Clinical samples of liver cancer and adjacent normal liver tissues were examined for malondialdehyde(MDA)contents and for expressions of mRNA and protein expressions of ACSL4 and proliferating cell nuclear antigen(PCNA)using RT-qPCR and Western blotting.Human liver cancer Huh-7 cells were treated with Erastin(a ferroptosis inducer),Fer-1(a ferroptosis inhibitor),or both,and the changes in expression levels of MDA,ACSL4 and PCNA were detected,and the cell proliferation was assessed with plate cloning assay.Results MDA contents were lower and ACSL4 and PCNA expressions were higher significantly in liver cancer tissues than in adjacent liver tissues.In Huh-7 cells,Erastin treatment significantly inhibited mRNA and protein expressions of ACSL4 and PCNA,suppressed cell proliferation,and increased MDA contents.Fer-1 alone did not produce significant effect on cell viability but reversed the effect of Erastin on ACSL4 and PCNA expressions,cell proliferation and MDA contents.Conclusion ACSL4 level is significantly overexpressed in liver cancer.Erastin increases MDA contents and down-regulates ACSL4 expression,thereby promoting ferroptosis and inhibiting proliferation of liver cancer cells,and these effects can be reversed by Fer-1.

Objective To investigate the clinical value of intermittent oral-to-esophageal tube feeding combined with perioral massage in treating dysphagia of children with Pierre Robin Syndrome (PRS). Methods A total of 92 PRS children with mandibular distraction osteogenesis were selected and divided into observation group and control group by stratified randomization, with 46 cases in each group. The control group received continuous nasal tube feeding intervention, while the observation group received intermittent oral-to-esophageal tube feeding combined with perioral massage intervention. The changes in nutritional status[body mass index (BMI), serum hemoglobin (Hb), total protein (TP), albumin (ALB)] before and after 1 month of intervention were compared between the two groups. The clinical indicators (hospital stay, time to start oral feeding, and time to full gastrointestinal feeding) were also compared 1 month after the intervention. The swallowing conditions and incidence of complications were recorded in both groups. Results After 1 month of intervention, BMI, Hb, TP and ALB in both groups significantly increased, and the observation group had significant higher values in BMI of (21.58±1.26) kg/m², Hb of (118.76±10.36) g/L, TP of (55.28±6.74) g/L, ALB of (33.46±4.37) g/L compared to BMI of (20.30±1.22) kg/m², Hb of (112.74±9.46) g/L, TP of (51.68±6.33) g/L and ALB of (30.59±4.68) g/L in the control group (P < 0.05). In the observation group, the feeding time was (32.26±4.43) minutes, hospital stay was (21.48±3.11) days, time to start oral feeding was (14.26±3.16) days, and time to full gastrointestinal feeding was (16.79±2.32) days, which were significantly shorter than (36.79±4.65) min, (24.39±3.31) days, (17.25±3.24) days, and (20.48±3.29) days respectively in the control group (P < 0.05); the average residual feeding volume was (10.26±2.18) mL and the number of cases with milk flow was 8 (17.39%) in the observation group, which were significantly less than (12.79±2.31) mL and 17 (36.95%) in the control group (P < 0.05). Conclusion Intermittent oral-to-esophageal tube feeding combined with perioral massage can effectively improve swallowing difficulty in children with PRS and shorten the hospital stay.