Intracytoplasmic sperm injection,which combined with in vitro fertilization and micromanipulation techniques,has been applied to research the molecular mechanisms of fertilization,and produce the sexing livestock and transgenic animals.The research advances in porcine intracytoplasmic sperm injection,including in vitro maturation and pretreatment of oocytes,selection and treatment of spermatozoon,artificial activation of injected oocytes after injection,and improvement of the operation technique were reviewed.

This study was aimed to collect relevant provisions of bulging disease medical records in the Ming and Qing dynasties to excavate the syndrome differentiation, drug law and medication frequency analysis. A database was established to analyze relevant provisions. The results showed that the syndromes of liver stagnation and spleen deficiency, dampness-heat accumulation, spleen-yang deficiency are the most common type. The mainly used drugs are from the category to eliminate dampness and water, and to tonify the deficiency. The top three used drugs are poria, atractylodes, and dried citrus peel. It was concluded that medications used in the Ming and Qing dynasties were mainly targeted to the disordered zangfu-organ, which was mainly about the spleen and the stomach. The syn-drome differentiation is the mixture of deficiency and excess. And deficiency is the main part. The medication should combine tonification and reducing. And tonification should be paid attention to. The syndrome differentiation and treatment should be coordinated and the primary and secondary aspect should be identified. These rules provide ref-erence effect for the clinical practice and scientific research of bulging disease treatment.

Objective To analyze the influencing factors of quality of life of patients with occupational pneumoconiosis in Methods Chongqing using a structural equation model. A total of 1 181 pneumoconiosis patients in Chongqing were selected - as the research subjects using convenient sampling method. The Chinese version of the SF 36 Scale was used to investigate the quality of life of the patients. Amos 24.0 software was used to construct a structural equation model reflecting the quality of life of Results ， （ ），（ ） （ ）， patients. The scores of physical health mental health and quality of life were 61.7±18.5 68.7±20.5 and 64.7±16.8 respectively. The scores of the eight dimensions of the quality of life of the research subjects were lower than that of the （ P ） Chongqing City municipal and national norms all <0.01 . Multiple linear regression results showed that personal monthly ， ， ， income pneumoconiosis stage complications employment status and smoking were the influencing factors of the quality of life （ P ） ， of pneumoconiosis patients all <0.05 . The results of the structural equation model showed that the stage of pneumoconiosis ， ， complications personal monthly income and pulmonary rehabilitation treatment could directly affect the physical and mental （ P ） ， health of the research subjects all <0.05 . In addition the stage of pneumoconiosis and complications indirectly affected the （ P ）Conclusion physical and mental health of patients by affecting pulmonary rehabilitation treatment all <0.05 . The structural equation model can be used to verify the influencing factors of the quality of life of patients with occupational pneumoconiosis. ， Complications personal monthly income and pneumoconiosis stage are the main factors affecting the quality of life of patients with occupational pneumoconiosis.

Objective To search for the changes of T cells subpopulations and immunoglobulin and their relation-ship in children patients with simple nephrotic syndrome. Design Case-control research. Patients aud Participants 39 patients with simple nephrotic syndrome were divided into two groups:the incipient group and relapse group (6 cases were determined at the incipient and relapse time) .Thereare 28 patients in incipient group, 19 males and 9 females, at the age of 2 to 10 years old. There are20 patients in relapse group, 12 males and 8 females, at the age of 3 to 13 years old. There are 35health children in control group, 21 males and 14 females, 2～13 years old. Interventions T cells subpopulations were determined by indirect immunofluorescence of OKT linesmonoclonal antibodies. The serum IgG was determined by routine simple agar immunodiffusion tests. Results and Conclusions The CD_3~+ and CD_4~+ cells are of no change in the children patients withsimple nephrotic syndrome, and the CD_8~+ and CD_(10)~+ cells are obviously increased, the Values of CD_4~+/CD_8~+ are obviously lower than those in the control qroup, there are no difference between the incipientand relapse groups. The levels of serum IgG were decreased in the 85.3% children patients, IgM were inc-reased in 29.4% of that. The values of CD_4~+/CD_8~+ have positive correlation and negative correlationwith the levels of serum IgG and IgM respectively.

Objective To study the role of protective antigen(PA)and N-terminal segment of lethal factor (LFn)in the entrance of EGFP(enhanced green fluorescent protein)into HeLa cells. Methods The DNA fragments encoding LFn and EGFP were amplified,respectively,and cloned into the plasmid pET-21 a(+)one after another to construct a recombinant plasmid pET-LFn-EGFP. The plasmid was txansformed into BL21 cells to express LFn-EGFP protein under the induction of IPTG. The protein was purified by Ni chelating chromatography. After incubation with LFn-EGFP in the presence of PA or not, the HeLa cells were analyzed by flow cytometry or laser confocal microscopy. Results The fusion protein LFn-EGFP was purified by over 90% homogeneity and retained the ability of LF to bind with PA when incubated with J774A.1 macrophage cells,and could get into HeLa cells. Conclusion The LFn-EGFP could enter the HeLa cells in a PA independent pathway. But PA could help more LFn-EGFP molecules enter into HeLa cells.

OBJECTIVETo observe the expression of phospholipid scramblase 1 (PLSCR1) in matrine (MAT) induced differentiation of all-trans retinoic acid (ATRA) resistant acute promyelocytic leukemia (APL) cells, and to explore its correlation to cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) signal pathway.

METHODSNB4 (an APL cell line sensitive to ATRA) and NB4-R1 (a resistant strain of ATRA) were observed as subjects in this study. Effects of combined treatment of 0.1 mmol/L MAT and 1 [mol/L ATRA on the differentiation of two cell lines were detected using nitroblue tetrazolium (NBT) reduction test and flow cytometry (CD11b). Expressions of PML/RARot and PLSCR1 protein/gene were detected using Western blot and Real-time fluorescence quantitative PCR assay. Meanwhile, H89, PKA antagonist, was used to observe cell differentiation antigen and changes of aforesaid proteins and genes.

RESULTSMAT combined ATRA could significantly elevate positive rates of NBT and CD11 b in NB4-R1 cells, and significantly down-regulate the expression of PML/RARapha-fusion protein/gene (P < 0.05, P < 0.01). ATRA used alone could obviously enhance the expression of PLSCRI in NB4 cells at protein and mRNA levels (P < 0.01). But the expression of PLSCR1 was up-regulated in NB4-R1 cells, but with statistical.difference only at the protein level (P <0. 01). In combination of MAT, PLSCR1 protein expression was further elevated in the two cell lines (P < 0.01). Besides, there was statistical difference in mRNA expressions in NB4-R1 cells (P < 0.05). All these actions could be reversed by treatment of 10 micromol/L H89 (P < 0.05, P < 0.01).

CONCLUSIONMAT combined ATRA could significantly induce the differentiation of NB4-R1 cells, and inhibit the expression of PML/RARalpha fusion gene/protein, which might be associated with up-regulating PLSCR1 expression.

Alkaloids ; Antineoplastic Agents ; Cell Differentiation ; Cell Line, Tumor ; Down-Regulation ; Humans ; Leukemia, Promyelocytic, Acute ; metabolism ; Phospholipid Transfer Proteins ; metabolism ; Quinolizines ; RNA, Messenger ; Signal Transduction ; Tretinoin ; Tumor Cells, Cultured ; Up-Regulation

Objective To establish a mouse model of prostate cancer expressing human PSCA for the development of new anti-tumor drugs or vaccines. Methods The total RNA of DU145 cells,a human prostate cancer cell line,was isolated by using TRIzol reagent according to the (RT-PCR),the first-strand cDNA was synthesized using the SuperScript First-Strand synthesis system. The human PSCA gene was amplified with the primers and cloned into the plasmid pcDNA3.1 to generate pcDNA-PSCA. DNA sequencing was used to confirm the constructs. The mouse prostate tumor cell line RM-1 cells,syngeneic to C57BL/6,were transfected with pcDNA-PSCA plasmids followed by selection using G418. RT-PCR analysis was performed to examine the validity of the constructs. Expression of PSCA on the cell surface was determined by staining with anti-PSCA antibody,and the anti-PSCA antibody was detected using an FITC-conjugated goat anti-rabbit IgG antibody,and analyzed by flow cytometry. 4-6-week-old male C57BL/6 mice purchased from the Laboratory Animals Center were inoculated with different amounts of RM-PSCA cells to search for suitable cell population which can form tumor in mouse,and the mice were monitored twice a week. The growth and the survival time of mice were measured,respectively. The tumor volume was measured by vernier caliper according to the formula:V=0.5a×b~2,where a and b are the long and short diameters of the tumor,respectively. Results The plasmid pcDNA-PSCA was successfully constructed and the PSCA was successfully expressed in RM-PSCA 7~# and RM-PSCA 28~# cells by RT-PCR and confirmed by flow cytometry. 1×10~5 RM-PSCA cells were sufficient to get tumor growth in 100% of inoculated mice. The tumor grew quickly and the volume of the tumor reached 12000 mm~3 within 34 days. All the mice died within 40 days and their mean survival time was 37 days. Conclusion A PSCA-expressing tumor model in mice has been successfully established. It can be used to evaluate the activities of drugs or vaccines.

Background and Objective:Histone deacetylase(HDAC) inhibitors can inhibit cell signal network function through decreasing expression of multiple genes and proteins,thus affect cell proliferation,survival and chemosensitivity.HDAC inhibitors combined with paclitaxel may enhance the inhibitory effect of drugs on lung cancer cells.This study was to observe the synergistic anti-proliferative effect of HDAC inhibitor trichostatin A (TSA)combined with paclitaxel on lung cancer cell lines H322 and H1299.and to investigate its mechanism. Methods:H322 and H1299 cells were divided into control group,paclitaxel(TAX) group,TSA group, and combination group(TF group,TSA followed by paclitaxel).Cell proliferation was determined by MTT assay.Cell cycle and apoptosis were determined by flow cytometry.The protein expression levels of survivin, ERK,and PARP were determined by Western blot analysis. Results: When combined with TSA,the 50%inhibition concentration(IC_(50))of paclitaxel decreased from (48.07 4±26.12) nmol/L to (6.34±5.72) nmol/L in H322 cells and from (110.64±38.7)nmol/L to(63.7±11.8)nmol/L in H1299 cells.with significant differences(P<0.05).Apoptosis rate of H322 cells was higher in the the TF group than in the TAX group(P<0.05).There were more necrosis cells in the TF group of H1299 cell line than in the other groups.pERK was up-regulated in the TAX group of H322 cell line.Expression of Survivin was up-regulated In the TAX group of two cells.Expressions of Survivin and pERK were downregulated in the TSA and TF groups of two cell lines. Cleaved PARP was detected in the TAX and the TF groups of H322 cells. and its expression was significantly higher in the the TF group than in the TAX group.Cleaved PARP was not detected in each group of H1299 cells.Conclusions:TSA combined with paclitaxel has a synergistic cytotoxicity effect on lung cancer cell lines H322 and H1299 when the cells were treated with TSA foIlowed by paclitaxel.The mechanism may be that TSA down-regulates the survivin highexpression induced by paclitaxel,and blocks pERK protein expression.

Nicotinic acetylcholine receptors (nAChRs) belong to ligand-gated ion channel receptors, of which α7 nAChR subtype is widely distributed in the cerebral cortex, thalamus, hippocampus, and also identified in microglia, macrophages, bone marrow cells, etc. Previous studies revealed that α7 nAChR is closely related to the function of the cholinergic anti-inflammatory pathway, and is a vital target for drug development of Alzheimer's disease and schizophrenia. The establishment of a stable α7 nAChR in vitro drug screening system is crucial for the efficient screening of novel drugs targeting this target. Recombinant expression of different subtypes of nAChRs on Xenopus laevis oocyte membranes and current detected by two-electrode voltage clamp (TEVC) is an advanced and complex model for novel drug screening. Molecular chaperones can assist the assembly of some nAChR subunits to form functional receptors, providing a stable expression model for the screening of compounds targeting this receptor. In this study, a molecular chaperone gene of α7 nAChR, transmembrane protein 35A (Tmem35a), was isolated and cloned from rats. We constructed the recombinant expression vector and obtained the cRNA of Tmem35a by in vitro transcription technique. Two cRNAs (Tmem35a and α7) were mixed and injected into X. laevis oocytes for expression. Then, the effects of this molecular chaperone on the current expression and pharmacological properties of α7 nAChR were evaluated by the TEVC. The results revealed that TMEM35A, also known as novel acetylcholine receptor chaperone (NACHO) could effectively increase the expression of α7 nAChR protein on oocyte membranes, and the amount of α7 nAChR protein was increased about 1-fold. The peak current induced by agonist acetylcholine (ACh) was increased about 10-fold. After injection of Tmem35a cRNA, the median effect concentration (EC₅₀) value of α7 nAChR to agonist ACh is 228.5 μmol·L^-1, which shows almost no difference from native α7 nAChR (EC₅₀: 223.3 μmol·L^-1), indicating the preservation of the normal properties of α7 nAChR. The results of this investigation indicate that the molecular chaperone NACHO effectively assists the heterologous expression of α7 nAChR in X. laevis oocytes, which provides a model for screening the potency of lead compounds targeting α7 nAChR. All animal experiments in this study were reviewed and approved by the Ethics Committee of Guangxi University (approval number: GXU-2023-0249).

N-type voltage-gated calcium (Ca²⁺) channels (N-type VGCC, Ca_V2.2) mediate Ca²⁺ influx in response to action potential at the presynaptic terminal, and play an important role in synaptogenesis, neurotransmitter release and nociceptive signal transduction. It is a new target for the development of drugs for the treatment of neuralgia (chronic pain) and other major diseases. Due to the difficulty of calcium channel expression in vitro and the detection of channel current, there is a great lack of new drug screening models. In this study, we established and optimized the electrophysiological drug screening model using Xenopus laevis oocytes for the recombinant expression of Ca_V2.2 in vitro (this study were reviewed and approved by the Ethics Committee of Guangxi University, approval number: GXU-2023-0249). Firstly, the linear plasmids encoding cDNA of major subunit α1B and auxiliary subunits α2δ1 and β3 of rat Ca_V2.2 were used as templates for in vitro transcription to generate their related mRNA (cRNA), after which three kinds of cRNA were injected into Xenopus laevis oocytes at the mass ratio of 2∶1∶1 for expression. The two-electrode voltage clamp (TEVC) technique was used to detect the inward current produced by Ca_V2.2. At the same time, the expression conditions of Ca_V2.2 were optimized, and its gating function was characterized from the aspects of channel activation and inactivation. The results showed that 3-5 days after cRNA microinjection, stable Ca_V2.2-mediated barium ion (Ba²⁺) currents were successfully detected. The interference of endogenous potassium channels and Ca²⁺-activated chloride channels can be eliminated by tetraethylammonium hydroxide (TEAOH) and 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis (BAPTA-AM) treatment. The maximum potential for Ca_V2.2 activation is 0 mV, and the current reverses to be outward when the membrane potential is greater than +50 mV. By fitting the steady-state activation and inactivation curves, the half-maximal activation potential and half-maximal inactivation potential of Ca_V2.2 are identified as -15.9 and -60.2 mV. In this study, a stable Ca_V2.2 expression system was established based on Xenopus laevis oocytes. The in vitro expression system can provide a new way for the screening of Ca_V2.2 active compounds or lead drugs.