Establishment of hepatocarcinoma cell line transfected by the B7 gene and its biocharacteristics

OBJECTIVE: To study the function of costimulation sign in tumor immunology and construct the new cell line B7^+ Smmc7721. METHODS: The B7 gene was transfected into the hepatocarcinoma cell Smmc7721 by liposma. Polymerase chain reaction (PCR) and reverse transcriptase-PCR (RT-PCR) were applied to test the result. MTT colorimetric assay was used to value the killing effect of lymphokine activated killer cells (LAK) activated by IL-2 on the transfected cell line and the original line. RESULT: B7^+ Smmc7721 was improved to be steadily expressed B7 molecule and LAK cells could more effectively act on the B7^+ Smmc7721 cells. CONCLUSION: The B7 gene can be transfected to hepatocarcinoma cells and can be expressed steadily in vitro, thus increasing the efficiency of LAK cells activated by IL-2 on them.

Biallelic mutations in spermatogenesis and centriole-associated 1 like(SPATC1L)cause acephalic spermatozoa syndrome and male infertility

Acephalic spermatozoa syndrome is a rare type of teratozoospermia that severely impairs the reproductive ability of male patients,and genetic defects have been recognized as the main cause of acephalic spermatozoa syndrome.Spermatogenesis and centrioleassociated 1 like(SPATC1L)is indispensable for maintaining the integrity of sperm head-to-tail connections in mice,but its roles in human sperm and early embryonic development remain largely unknown.Herein,we conducted whole-exome sequencing(WES)of 22 infertile men with acephalic spermatozoa syndrome.An in silico analysis of the candidate variants was conducted,and WES data analysis was performed using another cohort consisting of 34 patients with acephalic spermatozoa syndrome and 25 control subjects with proven fertility.We identified biallelic mutations in SPATC1L(c.910C>T:p.Arg304Cys and c.994G>T:p.Glu332X)from a patient whose sperm displayed complete acephalia.Both SPATC1L variants are rare and deleterious.SPATC1L is mainly expressed at the head–tail junction of elongating spermatids.Plasmids containing pathogenic variants decreased the level of SPATC1L in vitro.Moreover,none of the patient’s four attempts at intracytoplasmic sperm injection(ICSI)resulted in a transplantable embryo,which suggests that SPATC1L defects might affect early embryonic development.In conclusion,this study provides the first identification of SPATC1L as a novel gene for human acephalic spermatozoa syndrome.Furthermore,WES might be applied for patients with acephalic spermatozoa syndrome who exhibit reiterative ICSI failures.

Effects of Vitrification on the Imprinted Gene Snrpn in Neonatal Placental Tissue

Objective:To investigate the effects of vitrification on the expression of the imprinted gene Snrpn in neonatal placental tissue.Methods:Neonatal placental tissue was collected from women with natural pregnancy(control group)and from women in assisted reproductive technology(ART)pregnancy group,following fresh and vitrified embryo transfer(fresh group and vitrified group,respectively).Snrpn mRNA expression and SNRPN protein levels in placental tissue from these three groups were assessed by real-time reverse transcription polymerase chain reaction and Western blot,respectively.DNA methylation in the Snrpn promoter region was analyzed by bisulfite-pyrosequencing.Results:The expression of Snrpn mRNA and SNRPN protein was found to be higher in placental tissue from the fresh and vitrified ART groups,compared to the control group.There was no significant difference in SNRPN gene or protein expression between the fresh and vitrified groups.DNA methylation at the Snrpn promoter region was not significantly different between these three groups.Conclusions:Human ART may alter the transcriptional expression and protein levels of the imprinted gene Snrpn.However,compared to other ART methods,vitrification may not aggravate or reduce this effect.Moreover,the altered expression of Snrpn is likely not directly related to DNA methylation of the Snrpn promoter region.

Vitrification versus Slow Freezing of Human Oocytes: Effects on Ultrastructure and Developmental Potential

Objective:This study compared spindles,cytoskeleton,and developmental potential between vitrified and slow-frozen oocytes using PolScope and electron microscopy.Methods:Oocytes were randomly divided into control,slow freeze-thaw,and vitrification freeze-thaw groups(0,1,and 3 h).PolScope was used to observe spindles,angle of spindles to the first polar bodies,surface areas of oocytes,and lining and outer ret of zona pellucida.Surfaces and ultrastructure of oocytes were observed by scanning electron microscopy and transmission electron microscopy.These measures were used to characterize the impacts of two freezing methods on the developmental capacity of human oocytes.Results:The visible frequency of spindle formation was 92.4%,56.4%,11.2%,24.8%,and 61.1%in control group,slow freeze-thaw group,and the three vitrification freeze-thaw groups(0,1,and 3 h),respectively.Compared to oocytes in the slow freeze group,the angle of the spindle to the first polar body in oocytes in the 3-h vitrification freeze-thaw group was less(37.3°vs.68°,P=0.023).No significant differences were observed between the surface area of oocytes,or lining and outer ret of oocyte zona pellucida between freeze-thaw in these same two groups.Microvilli appeared normal.However,protrusions on oocyte surfaces were increased,and microvilli were laid down on the membrane surface in the 3-h vitrification freeze-thaw group in comparison to the slow-freeze group.Similar comparisons showed better recovery of perivitelline space and mitochondria between the 3-h vitrified and slow-frozen groups.Bipronuclear(2PN)fertilization rate observed in the slow-freeze group(65.7%)was lower than the rate seen in controls(79.2%,P=0.041).No significant differences were observed in 2PN fertilization,cleavage,and blastocyst formation rates between the 3-h vitrification freeze-thaw and control groups.Conclusions:Results suggest that vitrification freeze-thaw for oocyte cryopreservation was a better choice than slow freeze-thaw.