ObjectiveTo humanize the variable region of the IL9-2G1 rabbit monoclonal antibody （IL9 2G1 mAb） and to evaluate its biological activity. MethodsThe humanized sequences of IL9-2G1 mAb were designed. The heavy chain variable region （VH） and light chain variable region （VL） of the antibody were humanized and cloned into vector backbones containing the constant region frameworks of human IgG1 and Ig Kappa subtypes. Protein purification was carried out using the Chinese hamster ovary cell （CHO） expression system. Enzyme-linked immunosorbent assay （ELISA） was used to qualitatively analyze the antigen-binding activity of the antibodies. After screening the optimal humanized antibody candidate， surface plasmon resonance （SPR） was performed to detect molecular affinity. Western blot was used to examine the effects of the rIL-9 group and the rIL-9 + hIL-9 mAb group on the phosphorylation of Janus kinase 1/signal transducer and activator of transcription 3 （JAK1/STAT3）， while ELISA was employed to assess the impact of the Th9 group and the Th9 + hIL-9 mAb group on interleukin-9 （IL-9） secretion. ResultsPurification and ELISA results showed that the heavy chain OH and light chain FL exhibited good compatibility and expression performance. SPR results indicated that the equilibrium dissociation constant （KD） of M501042-OHFL against the analyte M30903 was 1.33×10^-7 M， which was superior to the parental 2G1 antibody. The Western blot results showed that the levels of phosphorylated JAK1 （p-JAK1） and phosphorylated STAT3 （p-STAT3） were significantly decreased in the rIL-9 + hIL-9 mAb group compared with the rIL-9 group （P<0.01）. The ELISA results indicated that the secretion of IL-9 was significantly reduced in the Th9 + hIL-9 mAb group compared with the Th9 group （P<0.01）. ConclusionHumanization modification is performed on the variable region of IL9-2G1 rabbit monoclonal antibody， and the humanized antibody hIL-9 mAb OHFL with high affinity and strong binding stability is successfully constructed， which provides an experimental basis for subsequent research.

ObjectiveTo obtain a construct chimeric antigen receptor macrophages （CAR-M） targeting disialoganglioside GD2 （GD2） and harboring the intracellular domain of macrophage-specific activating signaling adaptor protein DNAX-activating protein 12 （DAP12）. MethodsA CAR molecule targeting GD2 with the DAP12 intracellular domain as the signaling module ［Anti-GD2 CAR （DAP12）］ was designed， synthesized， and cloned into an adenoviral vector. This virus was used to infect PMA-differentiated THP-1 macrophages to construct green fluorescent protein （GFP）-labeled Anti-GD2 CAR THP-1 cells. CAR expression was confirmed using live-cell imaging. In vitro co-culture assays were performed to evaluate the phagocytic function of empty， Anti-GD2 CAR-M （CD3ζ）， and Anti-GD2 CAR-M （DAP12） groups against glioma cells with high （T98G） and low （U251） GD2 expression. ResultsThe CAR adenovirus efficiently transduced THP-1 macrophages， resulting in successful generation of Anti-GD2 CAR-M. Flow cytometry analysis following co-culture with glioma cells showed that compared to the empty group， the Anti-GD2 CAR-M （CD3ζ） group exhibited significantly enhanced phagocytosis （P<0.05）. Furthermore， compared to the Anti-GD2 CAR-M （CD3ζ） group， the Anti-GD2 CAR-M （DAP12） group demonstrated a further increase in phagocytic capacity specifically against GD2-high T98G cells （P<0.05）. ConclusionAnti-GD2 CAR-M （DAP12） cells are successfully established by transducing PMA-differentiated THP-1-M with recombinant Anti-GD2 CAR （DAP12） adenovirus. The engineered CAR-M can specifically target phagocytose glioma cells with high GD2 expression.

ObjectiveTo obtain stably transfected chimeric antigen receptor macrophages （CAR-M） targeting disialosylganglioside （GD2） based on the constitutive expression of myeloid differentiation factor 88 （MYD88）. MethodsAn Anti-GD2 CAR adenoviral vector incorporating the CD3ζ （cluster of differentiation 3 zeta chain）-MYD88 fusion construct was designed and constructed. THP-1-derived macrophages （THP-1M）， induced by phorbol 12-myristate 13-acetate （PMA） differentiation， were infected with this adenovirus to generate Anti-GD2 CAR THP-1 cells bearing a green fluorescent protein （GFP） tag. CAR expression was monitored using a live-cell imaging system. The phagocytic function of Empty CAR-M， Anti-GD2 CAR-M（CD3ζ）， and Anti-GD2 CAR-M（CD3ζ-MYD88） groups against glioma cells was evaluated in vitro through co-culture experiments， and their polarization status was also analyzed. ResultsCAR adenovirus successfully transfected THP-1 to obtain Anti-GD2 CAR-M. After co-culture with glioma cells， flow cytometry results showed that compared with the Empty CAR-M group and the Anti-GD2 CAR-M（CD3ζ） group， the Anti-GD2 CAR-M（CD3ζ-MYD88） group exhibited enhanced phagocytosis against T98G cells with high GD2 expression （P<0.05） and also showed upregulation of CD86 （P<0.05）. Compared with the Empty CAR-M group and the Anti-GD2 CAR-M（CD3ζ） group， the Anti-GD2 CAR-M（CD3ζ-MYD88）CAR-M group exhibited enhanced phagocytosis against T98G cells with high GD2 expression （P<0.05） and also showed upregulation of CD86 （P<0.05）， and the expression of inflammatory cytokines interleukin （IL）-6 and IL-12 was increased in this group （P<0.05）. ConclusionThis study successfully constructed Anti-GD2 CAR-M cells and verified their phagocytic activity， demonstrating that constitutive expression of MYD88 can promote inflammatory polarization of CAR-M. This provides a potential structural modification approach for the development of pro-inflammatory CAR-M.

Objective To verify the safety and effectiveness of a new percutaneous controllable curved plasma radiofrequency instrument for nucleus pulposus ablation. Methods A new percutaneous controllable curved plasma radiofrequency instrument were designed (controllable curved group), and its ablation effect was compared with the currently used straight head non-bendable plasma ablation instrument (non-bendable group) on gross specimens. The ablation instrument was placed through the right intervertebral foramen, and continuous ablation on the same intervertebral disc was conducted for three times. The ablation range and trajectory were recorded, and the temperature changes in the front, back, left, and right of the ablation center during and 15 seconds after ablation were monitored by the inserted temperature probe. Results There were no difference in temperature changes in the front, back, right regions of the ablation center during and 15 seconds after ablation between the two groups. The temperature changes in the left region of the ablation center both during and 15 seconds after 3rd ablation were larger than those in the non-bendable group (P＜0.01). Compared with the non-bendable group, the controllable curved group achieved angle control and larger single ablation area (2.282 5 mm² vs 1.135 8 mm², P＜0.000 1). Conclusions This new percutaneous controllable curved plasma ablation instrument can achieve angle control and ablation on the side opposite to the puncture site, increase ablation volume, and is safe.

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 observe the efficacy and safety of moxibustion in treating patients with central obesity of phlegm-dampness constitution. METHODS: A total of 66 patients with central obesity of phlegm-dampness constitution were randomly assigned to a moxibustion group (n=33, 3 cases dropped out) and a sham moxibustion group (n=33, 4 cases dropped out). The moxibustion group received mild moxibustion combined with lifestyle intervention; the moxibustion was applied at Shenque (CV8) and bilateral Zusanli (ST36), 30 min per session, maintaining a local skin temperature of (43±1) ℃. The sham moxibustion group received simulated moxibustion combined with lifestyle intervention; the simulated moxibustion was applied at the same acupoints, with the same session length, but with a maintained skin temperature of (37±1) ℃. Both groups were treated once every other day, three times per week for 8 consecutive weeks. Obesity-related physical indicators (waist circumference, hip circumference, body weight, body fat percentage, body mass index [BMI]), constitution evaluation indicators (phlegm-dampness constitution conversion score, symptom score), the impact of weight on quality of life-lite (IWQOL-Lite), the hospital anxiety and depression scale (HADS), and the incidence of adverse events were measured before and after treatment, and after 4 weeks of follow-up. RESULTS: Compared with before treatment, both groups showed significant reductions in waist circumference, hip circumference, body weight, body fat percentage, BMI, phlegm-dampness constitution conversion score and symptom score, IWQOL-Lite, and both anxiety and depression subscale scores of HADS after treatment and at follow-up (P<0.001). These improvements were significantly greater in the moxibustion group than those in the sham moxibustion group (P<0.001, P<0.01, P<0.05). One patient in the moxibustion group experienced a mild burn that resolved with routine care; the incidence of adverse reactions was 3.0% (1/33) in the moxibustion group and 0% (0/33) in the sham moxibustion group, with no statistically significant difference (P>0.05). CONCLUSION On the basis of lifestyle intervention, moxibustion effectively improves obesity-related physical indicators, enhances quality of life, alleviates anxiety and depression, and improves the phlegm-dampness constitution in patients with central obesity. These benefits persist for at least 4 weeks after treatment.
Humans ; Moxibustion ; Male ; Female ; Middle Aged ; Adult ; Obesity, Abdominal/psychology* ; Acupuncture Points ; Treatment Outcome ; Aged ; Quality of Life ; Young Adult ; Body Mass Index

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 study the plasma metabolomics of mice poisoned by different dosage of the combination of diazepam and ethanol,and to reveal the toxicological mechanisms of combined poisoning of diazepam and ethanol.Methods Female Kunming mice were randomly divided into blank group,single and combined poisoning group(n=6),Based on the LD50 of diazepam co-administered with graded ethanol doses,mice in the single-drug and combined groups received oral gavage at 1/2,1,and 2 × LD50.Retro-orbital blood samples(～500 μL)were collected within 24 hours post-administration and analyzed by UPLC-QE-MS technology.Principal component analysis and orthogonal partial least squares discriminant analysis were used to identify differential metabolites and associated metabolic pathways.Results A total of 387 differential metabolites were identified in the combined poisoning group of diazepam and ethanol implicating the key pathways including tryptophan metabolism,phenylalanine metabolism,arginine and proline metabolism,Glycerophospholipid metabolism,phenylalanine,tyrosine and tryptophan biosynthesis.Conclusion Combined diazepam and ethanol poisoning exerts significant systemic effects by disrupting neurotransmitters conduction,exacerbating oxidative stress response and dysregulating energy metabolism.