Host cell proteins(HCPs) of biological products are endogenous host proteins expressed by host cells or produced when they die during the production of biological products. Incomplete elimination of HCPs is the main factor causing allergy or other adverse reactions. The analytical methods for HCPs mainly include SDS-PAGE, Western blot(WB), two-dimensional gel electrophoresis(2DE), ELISA, liquid chromatography, mass spectrometry and so on. Electrophoresis can provide qualitative and semi-quantitative determination for proteins, but the resolution and accuracy are low. ELISA has good reproducibility and high sensitivity, and is most widely used at present. Chromatography and mass spectrometry have high separation and resolution as well as accurate quantification, but high application cost. ELISA was used to determine HCPs in recombinant biological products in the general rules <3412>, <3413> and <3414> of Chinese Pharmacopoeia(Volume Ⅲ, 2020 edition)and United States Pharmacopoeia(USP) <1132>. Analysis of HCPs components is helpful to improve the coverage of antiHCPs antibody and the sensitivity and specificity of detection methods. Traditional analytical methods can only qualitatively analyze HCPs, but cannot perform quantitative determination and characterization. Based on electrophoresis technology and mass spectrometry technology, combined with the development trend of HCPs component analysis at home and abroad, this paper summarizes the relevant HCPs analysis method to provide a reference for the verification of HCPs in the research and production process of biological products.

[Abstract ] In recent years, induced pluripotent stem cells (iPSCs) technology has played an important role in basic and clini-cal application research of Parkinson′s disease ( PD) and acquired significant progress .The neural progenitor /stem cells or dopamine ( DA) neurons which were obtained through iPSCs technique and direct differentiation technique from somatic cells were used for the study of cell therapy in PD , and good results were achieved .The cell models of DA neurons were established from PD patients carrying LRRK2, PAKK2, PINK or SNCA mutations via iPSCs technology , and the mitochondrial function and morphology , oxidative stress,α-synuclein ( SNCA) accumulation , and other aspects were studied on the pathogenesis of PD .This article briefly reviews the latest pro-gress of iPSCs technology in transplantation for treatment of PD and the establishment of cell model of PD disease , and provides refer-ence for further research .

Objective To investigate the influence of prepubertal exposure to estradiol benzoate (EB)in the male reproductive system of the rats and the natural process of tissue repair,and to clarify the possible mechanism of the reproductive toxicity of exogenous estrogen.Methods Ninety 2 1-day-old male Wistar rats were randomly divided into 2 experimental groups (low dose of EB group and high dose of EB group,n=30)and control group (n=30). The rats in the experimental groups were injected with EB dissolved in peanut oil at 15(low dose of EB group)and 15 000μg·kg-1 (high dose of EB group)respectively,the rats in control group received equal vehicle injection only,once every other day for two weeks from postnatal day(PND)21 to 34.All of them were normally fed after the drug usage was stopped.The testes were harvested at the stages of PND 60 and PND 125(n=15 at each stage).The serum levels testosterone of (T),follicle-stimulating hormone(FSH),luteinizing hormone(LH),prolactin (PRL)and estradiol(E2)of the rats in various groups were detected with radioimmunology method and the weights of the rats in various groups were recorded;the histological changes of the testes tissue were observed with light microscope.Results On PND60,compared with control group,the T levels in low dose of EB group and high dose of EB group were decreased(P<0.05 or P<0.01);the FSH,LH and E2 levels were increased (P<0.05 or P<0.01),and the PRL levels had no change(P>0.05);the weights of testes were decreased(P<0.01);the histological changes of the testes of the rats in experimental groups included seminiferous tubules maldevelopment, decreased cell number of seminiferous epithelia.Compared with low dose of EB group,the T and FSH levels in high dose of EB group were decreased (P<0.05 or P<0.01),the E2 and LH levels were increased(P<0.01),the PRL level had no change (P>0.05 ), and the weight of testes was decreased (P<0.01 );the diameters of seminiferous tubules were smaller,there was no sperm in high dose of EB group while there were a few sperms in low dose of EB group.On PND125,compared with control group,the T,FSH and PRL levels in low dose of EB group and high dose of EB group were decreased(P<0.01),the E2 levels were increased (P<0.01);the LH level in low dose of EB group was increased(P<0.05),the LH level in high dose of EB group was decreased(P<0.01), and the weights of testes in high dose of EB group were decreased(P<0.01);the diameters of seminiferous tubules and the cell number of seminiferous epithelia were increased but not apparent change.Compared with low dose of EB group,the T,LH levels in high dose of EB group were decreased (P<0.01),the E2 and FSH levels were increased(P<0.01),the PRL level had no change(P>0.05),and the weight of testes was decreased(P<0.01);there was still no sperm in high dose of EB group, the number of sperms was increased in low dose of EB group, but it was still lower than that in control group.Conclusion EB is harmful to the reproductive system and can change the normal serum sex hormone levels,even induces the irreversible injury.

Objective To investigate the effect of Sirt1 gene knockout on chronic kidney disease induced by 5/6 nephrectomy in mice and vascular endothelial growth factor (VEGF)/fetal liver kinase-1 (Flk-1) signaling pathway.Methods Twenty four male Sirt1 +/+ and Sirt1 +/-mice wererandomly divided into four groups:Sirt1+/+ mice with sham-operation (WT-Sham,n=6),Sirt1+/-mice with sham-operation (KO-Sham,n=6),Sirt1 +/+ mice with 5/6 nephrectomy (WT-Nx,n=6) and Sirt1 +/-mice with 5/6 nephrectomy (KO-Nx,n=6).Proteinuria was determined by urine collection from 8:00 to 8:00 the next day at 20 weeks.Serum creatinine (Scr),urea nitrogen (BUN) and the renal pathological changes were measured after 20 weeks.Expressions of Sirt1,collagen Ⅰ and transforming growth factor β(TGF-β) were used to analyze the changes of renal fibrosis by immunohistochemistry staining.Real-time PCR and Western blotting were used to measure the mRNA and protein expressions of Sirt1,fibronectin,collagen Ⅰ,VEGF and Flk-1 in kidney.Results Sirt1 expressed in glomernlar endothelial cells,podocytes,mesangial cells and renal tubular epithelial cells in Sirt1 +/+ mice,while Sirt1 expression intensity was significantly reduced in Sirt1 +/-mice.Compared with the WT-Sham group,WT-Nx group had increased proteinuria,BUN,Scr,glomernlar sclerosis index and tubulointerstitial fibrosis index at 12 weeks after operation (all P ＜ 0.01),and KO-Nx group had exacerbated the above up-regulations (all P ＜ 0.01).Compared with those in WT-Sham group,the expressions of fibronectin,collagen Ⅰ and TGF-β were up-regulated in WT-Nx group (all P ＜ 0.01),and were significantly augmented in KO-Nx group (all P ＜ 0.01).Compared with those in WT-Sham group,renal mRNA and protein expressions of VEGF and Flk-1 were decreased in WT-Nx group,and KO-Nx group aggravated their down-regulation (all P ＜ 0.01).Conclusions Sirt1 gene knockout can increase proteinuria and Scr,and aggravate renal pathology and renal fibrosis in 5/6 nephrectomized mice,which is associated with the inhibition of VEGF/Flk-1 signaling pathway.It is suggested that Sirt1 may be a potential therapeutic target of chronic kidney disease.

Objective:To detect the expression of tumor necrosis factor alpha in (TNF alpha) breast cacner and its relationship with imaging features.Methods:Using immunofluorescence and flow cytometry to detect the expression of TNF-α in MDA-MB-231 and MCF-7.Collect 82 patients with mammary gland disease,which was confirmed by pathological tissue,its pathological data,imaging data,and by immunohistochemistry to detect the expression of TNF-α in breast tissues,and analyze and the relationship between its expression and the pathological features and imaging characteristics.Results:TNF-α high expression in MDA-MB-231,the expression of TNF-oα in malignant breast tumor tissue significantly higher than that in benign tumor,the expression quantity associated with lymph node metastasis,TNM stages,strengthen uniform in MRI,the boundary and the shape of the X-ray Mammography (P=0.01),and color flow signal strength in ultrasound (P＜0.05).Conclusions:TNF alpha in breast tumor tissue was unusually high expression,and is closely related to some of the imaging features of breast tumor.

Objective To explore the effect of low-dose ketamine combined with propofol anesthesia on expres?sion of glutamine receptor subunit 1 (GluR1) and 2 (GluR2) in the hippocampus of depressed rats after electroconvulsive therapy. Methods Healthy adult male Sprague-Dawley rats, weighing 200~250 g, were used in this study. Mental depres?sion was induced by chronic unpredictable mild stress. Thirty-two depressed rats were randomly divided into 4 groups (n=8): metal depression group (group A), ECT group (group B), ECT+propofol group (group C) and ECT+propofol+ket?amine group (group D). Eight normal rats served as control group. Control group received no treatment. Group A received intraperitoneal injection of normal saline 8 mL/kg plus sham ECT. Group B, C and D received ECT once a day for 7 con?secutive days following intraperitoneal injection of normal saline 8 mL/kg, propofol 80 mg/kg and propofol 80 mg/kg +ketamine 10mg/kg, respectively. Sucrose preference test and Morris water maze were performed to assess depressed be?havior and learning and memory function, respectively. RT-PCR and Western-blot assay were used to detect the expres?sion of GluR1 , GluR2 and their mRNA expression. Results After ECT, compared with control group and group A, changes of SPP in group B, C and D were obvious. The change of SPP in group D was much higher than all other groups (P<0.05). Rats in group B showed prolonged escape latency and shortened space exploration time, which were significantly different from all other groups (P<0.05). Rats in group D showed the most shortened escape latency and prolonged space exploration time (P<0.05). The expression of GluR1 was significantly increased in group B, C and D compared with group A (P<0.05). The expression of GluR2 and mRNA was significantly decreased in group B and C (P<0.05). The difference in GluR2 and mRNA expression was not significant among group A, D and control group (P>0.05). Conclusion Low-dose ketamine combined with propofol anesthesia exert effective antidepressive action and improve learning and memory function of depressed rats after electroconvulsive therapy. The beneficial effects of the ketamine combined with propofol anesthesia may be related to up-regulation expression of GluR1 and GluR2 in hippocampus.

Objective To evaluate the effect of electroconvulsive therapy (ECT) on the expression of phosphorylated glutamate receptor 1 (p-GluR1) and Ca2+/calmodulin-dependent protein kinase Ⅱ α (p-CaMK Ⅱ α) under small dose ketamine combined with propofol anesthesia in the depressed rats.Methods Forty healthy adult male Sprague-Dawley rats,weighing 200-250 g,aged 2-3 months,were used in this study.Mental depression was induced by exposing the animals to chronic unpredictable mild stress (CUMS).Forty mentally depressed rats were divided randomly into 5 groups (n =8 each) using a random number table:M0-4 groups.Propofol 80 mg/kg and ketamine 10 mg/kg were injected intraperitoneally in M0-4 groups.After disappearance of righting reflex,M1-4 groups received ECT of 60,120,180 and 240 mC once a day for 7 consecutive days,respectively,by means of a current (frequency 50 Hz,sine-wave,pulse width 0.7 ms,1-s duration) delivered via ear-clip electrodes,while group M0 received ECT of no quantity of electric charge via ear-clip electrodes.Before CUMS,at 1 day after CUMS and at 1 day after ECT,sucrose preference test was applied to evaluate the depressive behavior.The sucrose preference percentage (SPP) was calculated.At 4 days after CUMS and 4 days after ECT,the learning and memory function was assessed using Morris water maze test.The rats were then sacrificed,and hippocampi were isolated to detect the expression of GluR1,p-GluRl,CaMK Ⅱ α and p-CaMK Ⅱ α by Western blot.Results The SPP was significantly lower after CUMS than before CUMS in M0-4 groups (P＜0.05).Compared with that after CUMS,the SPP was significantly increased,the escape latency was shortened,and the space exploration time was prolonged after ECT in M1-4 groups (P＜0.05).There was no significant difference in SPP after ECT between M1-4 groups (P＞0.05).Compared with group M0,the SPP was significantly increased,and the expression of pGluR1 and p-CaMK Ⅱ α was up-regulated in M1-4groups (P＜0.05).Compared with group M2,the escape latency was significantly prolonged,the space exploration time was shortened,and the expression of pGluR1 and p-CaMK Ⅱ α was down-regulated after ECT in the other groups (P＜0.05).There was no significant difference in GluR1 and CaMK Ⅱ α expression after ECT between the five groups (P＞ 0.05).Conclusion ECT can induce cognitive decline when applied for anti-depression under small dose ketamine combined with propofol anesthesia,and the mechanism is related to increased phosphorylation of GluR1 and CaMK Ⅱ α expression in rats.

Objective To investigate the expressions of fatty acid-binding protein 5 (FABP5)and dihydroli-poamide dehydrogenase(DLD)in skin lesions of symmetrical acrokeratoderma(SAK), and to explore their significance. Methods Biopsy specimens were obtained from skin lesions on the wrists and perilesional skin of 9 patients with SAK, and from normal skin in the wrists of 9 healthy volunteers (control group). Reverse transcription PCR (RT-PCR)and immunohistochemical staining were performed to measure the expressions of FABP5 and DLD in these specimens. Results RT-PCR showed no significant differences in the mRNA expressions of FABP5 or DLD between lesional, perilesional and normal control skin specimens(both P > 0.05). Immunohistochemically, there was a significant increase in the extent and intensity of staining for FABP5 in SAK lesions. Concretely speaking, FABP5 was strongly expressed in the stratum corneum, granular and spinous layers in SAK lesions, but weakly expressed in the stratum corneum, granular and spinous layers in perilesional skin, and only in spinous and basal layers in normal control skin. The expression of DLD decreased in SAK lesions, and was observed only in the stratum corneum and spinous layer in a few cases of SAK. However, the full-thickness epidermis stained positive for DLD in perilesional skin, with the nuclei and cytoplasm both stained deep brown. Conclusion The overexpression of FABP5 in SAK lesions may participate in dysdifferentiation of keratinocytes, while the down-regulation of DLD expression suggests an imbalance in energy metabolism.

Objective To observe the effect of rehabilitation nursing on activities of daily living (ADL) of patients with stroke in Midway-training Course. Methods 51 stroke patients were divided into control group (n=26) and supervision group (n=25). The control group received routine rehabilitation service, and the supervision group received rehabilitation nursing supervisor in addition. They were assessed with Barthel Index (BI) before and after the course. Results The BI increased after course in both groups (P<0.05), and increased in the supervision group (P<0.05) after 30 d, but not in the control group (P>0.05). Conclusion Rehabilitation nursing during Midway- training Course can improve recovery of the ADL of patients with stroke.

Objective : To evaluate the safety and immunogenicity of a split-virion quadrivalent influenza vaccine. Methods : The healthy people aged three years or over in Wuyang County and Xiping County of Henan Province were divided into the experimental group, control group 1 and control group 2, and were vaccinated with split-virion quadrivalent influenza vaccines, split-virion trivalent influenza vaccines (without B/Victoria) and a split-virion trivalent influenza vaccines (without B/Yamagata) , respectively. The hemagglutination inhibition (HI) antibodies were detected before and after immunization. The incidence rate of adverse events following immunization (AEFI) , HI antibody positive conversion rate, the protection rate of HI antibodies and the growth of geometric mean titer (GMT) were calculated and compared with the standard of Food and Drug Administration (FDA). Results: Totally 2 924 people were recruited, with 975 in the experimental group, 974 in the control group 1 and 975 in control group 2. The incidence rate of AEFI in the experimental group was 11.7%, higher than 7.9% in control group 1 and 8.8% in control group 2 (P < 0.05) during 30 minutes and 8 days after inoculation. The positive conversion rates of HI antibodies of H1N1, H3N2, By and Bv in the experimental group were 78.5%, 53.3%, 78.3% and 62.9%, respectively. The rate differences of the positive conversion rates of HI antibodies of By between the experimental group and control group 2, and of Bv between the experimental group and control group 1 were 42.1% (95%CI: 38.0%-46.2%) and 33.2% (95%CI: 28.9%-37.5%) , with both lower limits of 95%CI more than -0.10. The GMT increase of HI antibodies was more than 2.5 times in the three groups. The protective rates of HI antibodies of H1N1, H3N2, By and Bv in the experimental group were 87.7%, 98.7%, 93.6% and 77.2%, respectively. The protective rates of HI antibodies of By in control group 2 and Bv in control group 1 were 71.1% and 51.0%, both lower than those in the experimental group (P < 0.05). Conclusions After the inoculation of the quadrivalent influenza vaccine, the positive conversion rates (>40%) , protection rates (>70%) and GMT increase (>2.5 times) of HI antibodies of H1N1, H3N2, By and Bv all meet the quality standards of FDA. The safety and immunogenicity of the quadrivalent influenza vaccine are not inferior to those of the trivalent influenza vaccine.