Objective To study the profiling and authentication of human cell lines in cell bank of our department using short tandem repeat (STR) loci and to analyze the situation of cell contamination and misjudgment. Methods Sixty-one human cells including cells collected and preserved by our cell bank and cells from the other departments were detected by the 16 STR loci-method. To analyze the cross contamina-tion between human cell lines, the results were aligned with profiles published by international cell banks. Isoenzyme detection was employed to authenticate the cell species when the STR signal can not be detected. Results Among the 61 cells, specific profiles were produced by 41 ceils and there was no cross contamina-tion. Thirty-six cells had the completely same STR profiles in 9 STR loci with the same cells preserved by ATCC or JCRB, while 5 cells have different profiling just in the vWA loci. The cells referred above can be recognized as correct cells; Eleven cells (18.0%) were the false cells. Among them, cancer cells of tongue named Tca8113 and cancer cell of liver named HHCC(changed to FHCC98 now) had the same profile with HeLa and HeLa S3 respectively; Two ceils both named HUT-102 have the completely different profiles with ATCC; The signal of 4 cells was not be detected, and all of them were determined as hamster cell lines by u-sing isoenzyme detection. Also 2 cells were identified to be mixed cells. Conclusion The phenomenon of cell misjudgment and cross contamination between cells is serious. Authentication of cell lines correctly, es-pecially for the re-authenticatian of domestic self-established cells, is very important for the guarantee of the reliability and reproducibility for scientific researches.

Objective To study the prevalence and genotype of human parvovirus B19 virus among blood products and plasma pools in China. Methods B19 DNA derived from 16 lots of factor Ⅷ concentrate produced by 4 manufactures and 10 lots of plasma pools were detected by nested PCR. Phylogenetic comparison of the partial B19 sequences obtained from positive samples were performed by direct sequencing. Results Twelve of sixteen lots of factor Ⅷ concentrate and all of ten lots of plasma pools were contaminated by B19 DNA. By comparing the partial B19 sequences,all the isolated viruses were genotype Ⅰ and their nucleotides were high conserved with homology of 98. 3%-100%. Conclusion B19 genotype Ⅰ DNA has been detected in high prevalence in factor Ⅷ concentrate and plasma pools. The genetic diversities were shown to be very low.

Objective To study the application of short tandem repeat (STR) profiling in quality control of human cell lines used for biological production. Methods The methods detecting 9 and 16 STR loci to identify human cell lines by PCR-capiilary electrophoresis were established respectively. Human cell lines, which were derived from many corporations and including diploid cell strains used for virus-vaccine production and 293 cell lines used for gene therapy products, were analyzed and compared by these two methods. Results The STR profiling methods used for authentication of human cell lines were established. Most of human diploid cell strains(20/21 ) used for virus-vaccine production from 13 corporations were iden-tiffed as the intended cells and no cross-contamination was found. However, one MRC-5 cells was identified as a false cell line and one MRC-5 had 3 alleles in D13S317 locus. For 12 strains of 293 cell lines, there were significant differences in STR profiling from different manufactures, which was likely be explained that the sources and gene modifications of these 293 cell lines are not well known and their genes are unstable during passage. Conclusion The STR profiling method has the advantages of high sensitivity and specifici-ty, and can be used for authentication of each of human cell lines for biological production.

OBJECTIVETo explore the role of CD4⁺ CD25⁺ Foxp3⁺ Treg/CD4⁺ IL-17A(+)Th17 cells and the related cytokines in Graves' ophthalmopathy.

METHODSBased on clinical activity scores (CAS), we divided patients with untreated Graves' ophthal- mopathy into active group (AGO group with CAS ≥ 3 (15 cases) and non-active group (NGO group) with CAS<3 (15 cases), with another 15 patients with untreated Graves' disease free of eye symptoms (GD group) and 15 normal subjects as controls. Peripheral venous blood Treg/Th17 cell ratio was determined using flow cytometry. RT-PCR was used to detect the mRNA expression levels of Treg-specific transcription factor Foxp3 and Th17-specific transcription factor RORγt. Enzyme-linked immunosorbent assay (ELISA) was used to detect the serum levels of Th17 cell-related cytokines (IL-17A, IL-23, and IL-6) and Treg-related cytokines (TGF-β, IL-10, and IL-35).

RESULTSCompared with the normal subjects, the patients in GD, NGO, AGO groups all showed significantly increased Th17 cell count (P<0.05), which was the highest in AGO group. RT-PCR results revealed significantly increased RORγt in GD, NGO, and AGO groups, also the highest in AGO group. Serum IL-17A, IL-23, and IL-6 levels all showed significant increments in GD, NGO, and AGO groups (P<0.05), especially in AGO group. Among the Treg-related cytokines, TGF-β and IL-35 levels decreased (P<0.05) but IL-10 increased significantly (P<0.05) in GD, NGO, AGO groups.

CONCLUSIONDecreased immunosuppressive capacity of Treg cells can be an important factor in the pathogenesis of Graves' ophthalmopathy. Th17 cells may also participate in the occurrence and progression of Graves' ophthalmopathy and can serve along with related cytokines as novel indicators of the disease activity. Impaired Treg/Th17 balance may importantly contribute to the occurrence of Graves' ophthalmopathy.

Cytokines ; immunology ; Enzyme-Linked Immunosorbent Assay ; Flow Cytometry ; Graves Ophthalmopathy ; immunology ; Humans ; Nuclear Receptor Subfamily 1, Group F, Member 3 ; T-Lymphocytes, Regulatory ; immunology ; Th17 Cells ; immunology

After freeze-drying, the ultrastructure of boar sperms was observed by optical and electron microscopy. The in vitro development ability of the sperm was also examined with intracytoplasmic sperm injection (ICSI). The rate of male pronuclear formation was (68.52%), for cleavage (59.17%) and for blastocyst formation (19.16%) of the trehalose group (0.2 mol/L), significantly higher than those of the 50 mmol/L EDTA group (64.59%, 56.26% and 15.62%) and the control group (35.36%, 52.33% and 8.60%) (P < 0.05). After storage for 60, 120 and 180 d at 4 degrees C, no significant difference in the in vitro development was observed (P > 0.05). The male pronuclear, cleavage and blastocyst formation after ICSI with freeze-dried spermatozoa incubated for 1 h was superior than those incubated for 2 h (P < 0.05). No significant differences in the structures after stored at 4 degrees C or -20 degrees C (P > 0.05) were observed between the trehalose group and EDTA group. The percent of B grade freeze-dried boar spermatozoa in the trehalose group was higher than that of the EDTA group (P < 0.05). Based on the ultrastructure observation, main cryogenic damage in freeze-dried boar spermatozoa was swelling, damage or rupture in the sperm acrosome, neck and tail.
Animals ; Freeze Drying ; Male ; Semen Preservation ; methods ; veterinary ; Sperm Injections, Intracytoplasmic ; veterinary ; Spermatozoa ; Swine ; Trehalose ; pharmacology

Objective To explore the role of CD4+CD25+Foxp3+Treg/CD4+IL-17A+Th17 cells and the related cytokines in Graves' ophthalmopathy. Methods Based on clinical activity scores (CAS), we divided patients with untreated Graves' ophthal-mopathy into active group (AGO group with CAS≥3 (15 cases) and non-active group (NGO group) with CAS<3 (15 cases), with another 15 patients with untreated Graves' disease free of eye symptoms (GD group) and 15 normal subjects as controls. Peripheral venous blood Treg/Th17 cell ratio was determined using flow cytometry. RT-PCR was used to detect the mRNA expression levels of Treg-specific transcription factor Foxp3 and Th17-specific transcription factor RORγt. Enzyme-linked immunosorbent assay (ELISA) was used to detect the serum levels of Th17 cell-related cytokines (IL-17A, IL-23, and IL-6) and Treg-related cytokines (TGF-β, IL-10, and IL-35). Results Compared with the normal subjects, the patients in GD, NGO, AGO groups all showed significantly increased Th17 cell count (P<0.05), which was the highest in AGO group. RT-PCR results revealed significantly increased RORγt in GD, NGO, and AGO groups, also the highest in AGO group. Serum IL-17A, IL-23, and IL-6 levels all showed significant increments in GD, NGO, and AGO groups (P<0.05), especially in AGO group. Among the Treg-related cytokines, TGF-βand IL-35 levels decreased (P<0.05) but IL-10 increased significantly (P<0.05) in GD, NGO, AGO groups. Conclusion Decreased immunosuppressive capacity of Treg cells can be an important factor in the pathogenesis of Graves' ophthalmopathy. Th17 cells may also participate in the occurrence and progression of Graves' ophthalmopathy and can serve along with related cytokines as novel indicators of the disease activity. Impaired Treg/Th17 balance may importantly contribute to the occurrence of Graves' ophthalmopathy.

Objective To explore the role of CD4+CD25+Foxp3+Treg/CD4+IL-17A+Th17 cells and the related cytokines in Graves' ophthalmopathy. Methods Based on clinical activity scores (CAS), we divided patients with untreated Graves' ophthal-mopathy into active group (AGO group with CAS≥3 (15 cases) and non-active group (NGO group) with CAS<3 (15 cases), with another 15 patients with untreated Graves' disease free of eye symptoms (GD group) and 15 normal subjects as controls. Peripheral venous blood Treg/Th17 cell ratio was determined using flow cytometry. RT-PCR was used to detect the mRNA expression levels of Treg-specific transcription factor Foxp3 and Th17-specific transcription factor RORγt. Enzyme-linked immunosorbent assay (ELISA) was used to detect the serum levels of Th17 cell-related cytokines (IL-17A, IL-23, and IL-6) and Treg-related cytokines (TGF-β, IL-10, and IL-35). Results Compared with the normal subjects, the patients in GD, NGO, AGO groups all showed significantly increased Th17 cell count (P<0.05), which was the highest in AGO group. RT-PCR results revealed significantly increased RORγt in GD, NGO, and AGO groups, also the highest in AGO group. Serum IL-17A, IL-23, and IL-6 levels all showed significant increments in GD, NGO, and AGO groups (P<0.05), especially in AGO group. Among the Treg-related cytokines, TGF-βand IL-35 levels decreased (P<0.05) but IL-10 increased significantly (P<0.05) in GD, NGO, AGO groups. Conclusion Decreased immunosuppressive capacity of Treg cells can be an important factor in the pathogenesis of Graves' ophthalmopathy. Th17 cells may also participate in the occurrence and progression of Graves' ophthalmopathy and can serve along with related cytokines as novel indicators of the disease activity. Impaired Treg/Th17 balance may importantly contribute to the occurrence of Graves' ophthalmopathy.

Objective:To evaluate the efficacy of ultrasound-guided semispinalis capitis plane (SCP) block for treatment of occipital neuralgia (ON).Methods:This was a prospective study. Ninety patients of both sexes, aged 29-66 yr, suffering ON for 3 months-6 yr in Zibo Municipal Hospital from January 2022 to December 2023, were divided into 3 groups ( n=30 each) using a random number table method: combination of greater occipital nerve (GON) block and the third occipital nerve (TON) block group (group GT), SCP block via the medial head of semispinalis capitis muscle (SCM) group (group Sm), and SCP block via the space between obliquus capitis inferior and C 2, 3 facet joint (OCI-C 2, 3) group (group OC). In GT group, the analgesic and anti-inflammatory compound solution 2.5 ml was injected around GON in the SCM-OCI space at the C 2 level of the cervical vertebra and at the lateral surface of C 2, 3 facet joint. In Sm group, the analgesic and anti-inflammatory compound solution 5 ml was injected into the medial head of SCM at the level of C 1. In OC group, the analgesic and anti-inflammatory compound solution 5 ml was injected into the OCI-C 2, 3 space in the deep part of SCM. The Visual Analogue Scale (VAS) score and Pittsburgh Sleep Quality Index (PSQI) score were recorded before treatment (T 1) and at 1, 3, 7, 10 and 14 days after treatment (T 2-6), and then the rates of pain relief and improvement in sleep quality were calculated. The time spent in blocking, onset time of blocking, completion time of blocking, duration of block, and occurrence of adverse reactions within 24 h after block were recorded. Results:There were no significant differences in VAS scores and PSQI scores at T 1-3 and T 5-6 among the three groups ( P>0.05), and VAS and PSQI scores were significantly higher at T 4 in Sm group than in OC and GT groups ( P<0.05). Compared with GT group, the time spent in blocking was significantly shortened, the onset time and completion time of block was prolonged, and the duration of block was shortened in Sm group, and the time spent in blocking was significantly shortened, the onset time and completion time of block was shortened ( P<0.05), and no significant change was found in the duration of block in OC group ( P>0.05). No severe complications were observed in the three groups. Conclusions:Compared with the combination of GON and TON blocks, ultrasound-guided SCP block for treating ON is simple and highly safe, SCP block via the OCI-C 2, 3 space has rapid onset and long duration, leading to significant improvements in pain and sleep quality, and it can be used as the first-choice block method for treating ON.