To knock out β-lactoglobulin (BLG) gene and insert human lactoferrin (hLF) coding sequence at BLG locus of goat, the transcription activator-like effector nucleases (TALEN) mediated recombination was used to edit the BLG gene of goat fetal fibroblast, then as donor cells for somatic cell nuclear transfer. We designed a pair of specific plasmid TALEN-3-L/R for goat BLG exon III recognition sites, and BLC14-TK vector containing a negative selection gene HSV-TK, was used for the knock in of hLF gene. TALENs plasmids were transfected into the goat fetal fibroblast cells, and the cells were screened three days by 2 μg/mL puromycin. DNA cleavage activities of cells were verified by PCR amplification and DNA production sequencing. Then, targeting vector BLC14-TK and plasmids TALEN-3-L/R were co-transfected into goat fetal fibroblasts, both 700 μg/mL G418 and 2 μg/mL GCV were simultaneously used to screen G418-resistant cells. Detections of integration and recombination were implemented to obtain cells with hLF gene site-specific integration. We chose targeting cells as donor cells for somatic cell nuclear transfer. The mutagenicity of TALEN-3-L/R was between 25% and 30%. A total of 335 reconstructed embryos with 6 BLG-/hLF+ targeting cell lines were transferred into 16 recipient goats. There were 9 pregnancies confirmed by ultrasound on day 30 to 35 (pregnancy rate of 39.1%), and one of 50-day-old fetus with BLG-/hLF+ was achieved. These results provide the basis for hLF gene knock-in at BLG locus of goat and cultivating transgenic goat of low allergens and rich hLF in the milk.
Animals ; Animals, Genetically Modified ; genetics ; Female ; Fibroblasts ; Gene Knock-In Techniques ; Gene Knockout Techniques ; Goats ; genetics ; Humans ; Lactoferrin ; genetics ; Lactoglobulins ; genetics ; Milk ; chemistry ; Nuclear Transfer Techniques ; Plasmids ; Pregnancy ; Transfection

To demonstrate the current strategies for treating cartilage defects of knees and the related research. Published papers about cartilage defects were searched and reviewed. The current strategies for the treatment were summarized. Based on the research of our study and others, the conclusion how to treat cartilage defects was made. The current ways for treating cartilage defects include micro-fractures, chondrocytes transplantation, mosaicplasty and tissue engineering; Research on functional magnetic resonance imaging in the early diagnosis of cartilage defects, cartilage degeneration is gradually increasing. There is still no effective treatment of cartilage defects and tissue engineering has brought new hopes for the treatment of cartilage defects , functional magnetic resonance imaging has some significance in early diagnosis of cartilage defects, cartilage degeneration.
Animals ; Cartilage Diseases ; surgery ; therapy ; Cartilage, Articular ; surgery ; Humans ; Knee ; surgery ; Tissue Engineering ; Transplantation, Autologous

Calcinosis ; diagnostic imaging ; etiology ; Child ; Child, Preschool ; Female ; Globus Pallidus ; pathology ; Humans ; Male ; Osteogenesis Imperfecta ; complications ; genetics ; Skull ; diagnostic imaging ; Tomography, X-Ray Computed

To investigate the clinicopathological features of endometrial endometrioid adenocarcinoma(EEA)with adnexal involvement.Methods The clinicopathological data of 86 EEA patients who underwent surgical treatment at our center from January 2000 to December 2015 were analyzed retrospectively.The clinicopathological features were compared between patients with occult adnexal involvement and those with gross adnexal involvement.Results A total of 86 EEA patients with adnexal involvement(mean age:58.1 years)were included in this study,accounting for 5.4%(86/1592)of the EEA patients during the same period.Among these 86 patients,there were 13 premenopausal patients(15.1%)including 2 premenopausal patients aged under 40 years.Gross adnexal involvement was found in 47 patients(54.7%),while occult adnexal involvement was found in 39 patients(45.3%)in pathology evaluation.Ovarian metastasis was found in 34 patients(39.5%),followed by both ovarian and tubal metastasis in 19 patients(22.1%)and tubal metastasis in 33 patients(38.4%).The expressionss of estrogen receptor(χ=8.086,P=0.042)and progesterone receptor(PR)(χ=9.149,P=0.026)were significantly different between gross adnexal involvement group and occult adnexal involvement group,whereas no significant difference was found in other clinicopathological features(all P>0.05).The non-conditional Logistic regression analysis showed that,compared with PR no-expression group,the rate of occult microscopic adnexal involvement in PR low-expression group was 6.375 times of that of the gross adnexal involvement(P=0.005,95%CI:1.768-22.976),and the rate of occult microscopic adnexal involvement in the PR high-expression group was 3.719 times of that of gross adnexal involvement(P=0.048,95%CI:1.009-13.702). Conclusion PR expression level is remarkably lower in EEA patients with gross adnexal involvement than those with occult adenxal involvement.

OBJECTIVETo screen and identify apoptosis related proteins and explore the mechanism of harringtonine (HT)-induced K562 cells apoptosis.

METHODSFlow cytometry was used to distinguish K562 cells in the earlier stage of apoptosis from those in the later stage of apoptosis by annexin V and PI staining. Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) coupled with computer image analysis was used to detect the changes in protein expression in the two stages of apoptosis. Proteins were identified by peptide mass fingerprint in combination with database searching.

RESULTSK562 cells treated with HT for 5 and 24 hours were in the early and later stages of apoptosis respectively. Statistical analysis showed 3 spots disappeared, 7 spots with decreased intensity and 10 spots with increased intensity in the 24 h HT induced apoptotic cells as compared with that in 5 h HT induced ones. Ten spots were selected on the basis of the intensity and the significant changes in abundance. Among them, 5 apoptosis related proteins were successfully identified by MALDI-TOF: keratin 9, BTF3, TrpRS, RS and prohibitin.

CONCLUSIONSUp-regulation of TrpRS, RS, prohibitin and down-regulation of BTF3 were involved in inhibition of transcription and protein synthesis in the apoptotic K562 cells induced by HT, whereas up-regulation of keratin 9 was related to apoptosis resistance.

Apoptosis ; drug effects ; Electrophoresis, Gel, Two-Dimensional ; Harringtonines ; pharmacology ; Humans ; K562 Cells ; drug effects ; metabolism ; pathology ; Proteome ; metabolism

Objective: To study the influence of electret on surface charge of fibroblast cells (3T3 cells) and to probe the relationship between cell growth, apoptosis and cell surface charge. Methods: Electrets Teflon PTFE, ±300 V,±1 000 V were used to treat 3T3 cells for 24, 48 and 72 h. Then the influences of electrets on cell cycle and surface charge of 3T3 cells were studied by flow cytometry and electrophoresis, respectively. Results: (1) After 24 h action of negative electrets, electrophoretic mobility (or surface charge) and cell number in S phase of 3T3 cells were significantly increased compared with those in control group. (2) Effect of negative electrets enhancing cell growth and increasing cell surface charge was in proportional to the surface potential of electret. (3) Surface charge density of apoptotic cell was reduced by electret. (4) After 24 h action of positive electret, the cell number in S and G2 phase were decreased and cell surface charge was also reduced. Conclusion: Negative electret can improve cell growth and increase cell surface charge density. Positive electret can restrain cell growth and reduce cell surface charge density. Surface charge of apoptotic cell is less than that of normal cell.

This study was designed to evaluate the effects of drilling through the growth plate and using adipose-derived stem cells (ADSCs) and bone morphogenetic protein-2 (BMP-2) to treat femoral head epiphyseal ischemic necrosis,which can be done in juvenile rabbits.Passage-four bromodeoxyuridine (BrdU)-labeled ADSCs were cultured,assayed with MTT to determine their viability and stained with alizarin red dye to determine their osteogenic ability.Two-month-old,healthy male rabbits (1.2 to 1.4 kg,n=45) underwent ischemic induction and were randomly divided into five groups (group A:animal model control;group B:drilling;group C:drilling & ADSCs;group D:drilling & BMP-2;and group E:drilling & ADSCs & BMP-2).Magnetic resonance imaging (MRI),X-ray imaging,hematoxylin and eosin staining and BrdU immunofluorescence detection were applied 4,6 and 10 weeks after treatment.Approximately 90％ of the ADSCs were labeled with BrdU and showed good viability and osteogenic ability.Similar results were observed in the rabbits in groups C and E at weeks 6 and 10.The animals of groups C and E demonstrated normal hip structure and improved femoral epiphyseal quotients and trabecular areas compared with those of the groups A and B (P＜0.01).Group D demonstrated improved femoral epiphyseal quotients and trabecular areas compared with those of groups A and B (P＜0.05).In summary,drilling through the growth plate combined with ADSC and BMP-2 treatments induced new bone formation and protected the femoral head epiphysis from collapsing in a juvenile rabbit model of femoral head epiphyseal ischemic necrosis.

OBJECTIVETo study the mechanical properties of Co-Cr ceramic alloy after recasts.

METHODSCo-Cr ceramic alloy cast samples were prepared and recast for 3 times without adding any new Co-Cr ceramic alloy. The tensile strength, 0.2% yield strength, percentage of elongation, flexural strength, flexural modulus and Vickers hardness of each specimen were measured.

RESULTSBeing cast for different times, the Co-Cr ceramic alloy showed no significant differences on their tensile strength, 0.2% yield strength, percentage of elongation, flexural strength, flexural modulus and Vickers hardness.

CONCLUSIONCo-Cr ceramic alloy can be recast for 3 times at least, without decrease of the mechanical properties.

Alloys ; Ceramics ; Chromium Alloys ; Dental Stress Analysis ; Hardness ; Materials Testing ; Metal Ceramic Alloys ; Tensile Strength

OBJECTIVETo observe the effects of prenatal exposure to low level lead on the protein and mRNA expression of growth-associated protein (GAP-43) in hippocampus of rat's offspring, and to explore the molecular mechanisms of lead on learning and memory.

METHODSThe pregnant rats were randomizedly divided into 4 groups and provided with doubly evaporated water in control group and 125, 250, 500 mg/L lead acetate solution via drinking water in treatment groups respectively during pregnancy. When the rat's offspring was 1, 21, 60 days old, the lead content in hippocampus was measured by hydride generation atomic absorption spectrometry, and the GFAP protein and mRNA expression at hippocampal CA1 region were observed by immunohistochemistry and in situ hybridization.

RESULTSThe content of lead in the hippocampus was (1.64 +/- 0.32), (2.33 +/- 0.42) and (3.28 +/- 0.58) microg/L, and (0.94 +/- 0.18), (1.27 +/- 0.26) and (1.79 +/- 0.42) microg/L respectively in the low, middle and high lead dosage group when the rat's offspring was one day and 21 day old. When the rat's offspring was 1, 21 days old, the content of lead in hippocampus in treatment groups was significant higher than that of control (P < 0.05), the integral optical density of GAP-43 protein and mRNA expression (except low dosage treatment at 21 d) were significantly decreased compared with the control (P < 0.01, P < 0.05), but there was no significant difference at 60-day old offsprings for the parameters above.

CONCLUSIONExposure to low level lead during pregnancy could inhibit the GAP-43 protein and mRNA expression in hippocampus of rat's offspring, which may be one of the molecular mechanisms of lead on learning and memory.

Animals ; Environmental Exposure ; adverse effects ; Female ; GAP-43 Protein ; genetics ; metabolism ; Hippocampus ; drug effects ; metabolism ; Lead ; toxicity ; Pregnancy ; Prenatal Exposure Delayed Effects ; metabolism ; RNA, Messenger ; genetics ; Rats ; Rats, Wistar

OBJECTIVEThymidylate synthase (TS) catalyses the conversion of deoxy-uridylate to deoxy-thymidylate and is a key enzyme for DNA synthesis. TS is the target enzyme of 5-fluorouracil (5-FU) and involved in folate metabolism. TS gene polymorphisms play an important role in the efficiency of fluorouracil activity in vivo. This study investigated the allelic frequencies and distribution characters of single-nucleotide polymorphisms within the coding region (cSNPs) of TS gene in Chinese children with acute leukemia (AL) and normal control children in order to explore the possible relationship between the cSNP in human TS gene and chemotherapeutic effects of 5-fluorouracils.

METHODSBone marrow samples from 53 children with AL and peripheral blood samples from 115 normal children were obtained to prepare complementary DNAs (cDNAs). The cDNAs were analyzed for the polymorphisms in TS gene by reverse transcriptase (RT)-polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE) and direct sequencing. The distributive difference of each genotype between AL children and control children was evaluated.

RESULTSA polymorphism 381 A>G (E127E) in the coding region of TS gene was firstly identified in the Chinese population. The 381 A>G allelic frequency in AL children and control children was 12.3% and 13.5% respectively (P>0.05), which were similar to that in the International SNP Bank (12.3%). The allelic frequency of cSNPs was not associated with the susceptibility to AL.

CONCLUSIONSA polymorphism 381 A>G (E127E) in TS gene was successfully identified in children using RT-PCR-DGGE combined with DNA sequencing. There was no significant difference in the allelic frequency of cSNPs in AL children and normal children.

Acute Disease ; Child ; Child, Preschool ; Electrophoresis, Polyacrylamide Gel ; Female ; Humans ; Infant ; Infant, Newborn ; Leukemia ; genetics ; Male ; Polymorphism, Single Nucleotide ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Sequence Analysis, DNA ; Thymidylate Synthase ; genetics