To synthesize KLD-12 peptide with sequence of AcN-KLDLKLDLKLDL-CNH(2) and trigger its self-assembly in vitro, to encapsulate rabbit MSCs within peptide hydrogel for 3-D culture and to evaluate the feasibility of using it as injectable scaffold for tissue engineering of IVD. KLD-12 peptide was purified and tested with high performance liquid chromatography (HPLC) and mass spectroscopy (MS). KLD-12 peptide solutions with concentrations of 5 g/L, 2.5 g/L and 1 g/L were triggered to self-assembly with 1xPBS in vitro, and the self-assembled peptide hydrogel was morphologically observed. Atomic force microscope (AFM) was employed to examine the inner structure of self-assembled peptide hydrogel. Mesenchymal stem cells (MSCs) were encapsulated within peptide hydrogel for 3-D culture for 2 weeks. Calcein-AM/PI fluorescence staining was used to detect living and dead cells. Cell viability was observed to evaluate the bioactivity of MSCs in KLD-12 peptide hydrogel. The results of HPLC and MS showed that the relative molecular mass of KLD-12 peptide was 1467.83, with a purity quotient of 95.36%. KLD-12 peptide at 5 g/L could self-assemble to produce a hydrogel, which was structurally integral and homogeneous and was able to provide sufficient cohesion to retain the shape of hydrogel. AFM demonstrated that the self-assembly of KLD-12 peptide hydrogel was successful and the assembled material was composed of a kind of nano-fiber with a diameter of 30-40 nm and a length of hundreds of nm. Calcein-AM/PI fluorescence staining revealed that MSCs in KLD-12 peptide hydrogel grew well. Cell activity detection exhibited that the A value increased over the culture time. It is concluded that KLD-12 peptide was synthesized successfully and was able to self-assemble to produce nano-fiber hydrogel in vitro. MSCs in KLD-12 peptide hydrogel grew well and proliferated with the culture time. KLD-12 peptide hydrogel can serve as an excellent injectable material of biological scaffolds in tissue engineering of IVD.

Genetic modification technology is a new molecular tool for targeted genome modification. It includes zinc finger nucleases (ZFN) technology, transcription activator-like effector nucleases (TALEN) technology and clustered regularly interspaced short palindromic repeat (CRISPR)-associated (Cas) (CRISPR-Cas) nucleases technology. All of these nucleases create DNA double-strand breaks (DSB) at chromosomal targeted sites and induce cell endogenous mechanisms that are primarily repaired by the non-homologous end joining (NHEJ) or homologous recombination (HR) pathway, resulting in targeted endogenous gene knock-out or exogenous gene insertion. In recent years, genetic modification technologies have been successfully applied to bacteria, yeast, human cells, fruit fly, zebra fish, mouse, rat, livestock, cynomolgus monkey, Arabidopsis, rice, tobacco, maize, sorghum, wheat, barley and other organisms, showing its enormous advantage in gene editing field. Especially, the newly developed CRISPR-Cas9 system arose more attention because of its low cost, high effectiveness, simplicity and easiness. We reviewed the principles and the latest research progress of these three technologies, as well as prospect of future research and applications.
Animals ; CRISPR-Cas Systems ; DNA Breaks, Double-Stranded ; Endonucleases ; Genetic Engineering ; methods ; Humans ; Mutagenesis, Insertional ; Mutagenesis, Site-Directed ; Plants ; Zinc Fingers

Objective To explore the applied value of urine light chainκ、λand κ/λ ratio test in older people with B cell malignant proliferative disease.Methods Young volunteers, general older patients, kidney failure older patients and older patients with B cell malignant proliferative disease were selected and immunoephe-lometry method was applied to detect the level of urine light chainκ、λ and κ/λ ratio.Result The average levels (mg/L)of urine light chain κand λin older patients with kidney failure group(172.00 ±188.10,111.50 ± 109.32)were higher than that in general older patients group(32.72 ±33.60,15.02 ±15.58).In each of the ol-der patients groups,the levels of urine light chainκandλwere higher than that in young volunteers groups(9.30 ±5.80,4.97 ±2.61).The κ/λ ratios of urine light chain in older patients with kidney failure group(1.59 ± 0.4),general older patients group(2.19 ±0.54)and young volunteers group(1.92 ±0.48)were consistent,how-ever,it was significantly abnormal in older patients with B cell malignant proliferative disease group,the ratio was high inκtype(44.8 ±83.17)and low inλtype(0.06 ±0.08).After effective treatment, κ/λ ratio of urine light chain in older patients with B cell malignant proliferative disease tended to normal.Conclusion The level of u-rine light chainκandλis effected by renal function,but not involved the κ/λ ratio.B cell malignant proliferative disease significantly affects theκ/λratio of urine light chain.Constantly monitoring the change ofκ/λratio of u-rine light chain in older peoples with B cell malignant proliferative disease can reflect the proliferative degree of malignant B cell in vivo.

Objective To explore the apoptosis effect induced by bortezomib combined with homoharringtonine or arsenious acid in HL-60 cell line and the mechanism.Methods Cell' s apoptosis was demonstrated by MTT assay and Hoechst33342 staining.Expression of bcl-2,Caspase-9,Caspase-3 and PARP protein was detected by Western blot.Results HL-60 cells' apoptosis could be induced by bortezomib,homoharringtonine and arsenious acid respectively.Proliferation inhibition of HL-60 cells could be enhanced significantly when treated by bortezomib combined with homoharringtonine or arsenious acid compared with treated by any of the three drugs alone (P ＜ 0.05).At the same time morphology shows the apoptosis induced by drugs combined is more obviously than by one drug.Western blot showed bcl-2 protein was down-regulated and Caspase-9,Caspase-3 and PARP proteins were all cleaved activation when cells were treated by 15 μmol/ L arsenious acid alone,but only cleaved activation of PARP and down-regulation of bcl-2 protein be detected when cells were treated with 30 nmol/L homoharringtonine alone,expression of Caspase-9 and Caspase-3 had no change compared with the control.The changes of associated proteins were paralleled with the cell' s apoptosis when treated with combined drugs.Conclusion HL-60 cells' apoptosis effect is inhanced significantly when bortezomib combined with homoharringtonine or arsenious acid.Arsenious acid and bortezomib can inhibit caspase signaling pathway and down-regulate the expression of bcl-2 protein together,but homoharringtonine and bortezomib can only down-regulate the expression of bcl-2 protein and induce cleaved activation of PARP together.

Objective To investigate the relationship between circulating tumor cells(CTCs) in peripher-al blood and the prognosis of pancreatic cancer patients. Method From Mar 2010 to Mar 2015, patients with pancreatic cancer in guangzhou first people′s hospital were enrolled in this study. Immunomagetic negative en-richhment together with immunofluorescence were used to identify CTCs. The clinical data of all patients were analyzed. Result No CTCs were found in peripheral blood of healthy control cases. The detection date of CTCs was 60.0%. The positive rate of CTCs was closely correlated with cell differentiation and the clinical stage. Con clusion CTCs count in old patients with pancreatic cancer can reflect the status of the patients and is help-ful for the diagnosis of micrometastasis , re-determination of the clinical stage and the guidance of patients′s treatment. CTCs count can predict the survival time of elderly patients with pancreatic cancer.

Objective To study the anti-tumor effect and mechanism of bortezomib in primary acute leukemia cells from elderly patients.Methods Primary acute leukemia cells were treated with bortezomib 50-5000 nmol/L for 24-48 h,cell proliferation was analysed by MTT assay; apoptosis of primary acute leukemia cells was observed by fluorescence microscopy and flow cytometry; protein expression of bcl-2 and Bax was detected by Western blot.Results The cell viability was 90 ％ and 70 ％ when leukemia cells were treated with 50 and 5000 nmol/L bortezomib for 24 h,respectively.Meanwhile,cells showed (10.2±2.3) ％ and (13.3±3.3) ％ apoptosis.With prolonged treatment for 48 h,cell viability decreased to 86 ％ and 60 ％,respectively,while the apoptosis rates were increased to(18.4±3.9) ％ and(20.7±3.7) ％.Compared to the control group 0 nmol/L bortezomib,the differences were statistically significant (F =53.76,F =7.74,F =54.49,F =16.94,all P values ＜ 0.05).With the increase of bortezomib concentration,the bcl-2 protein expression was decreased,while Bax was up-regulated.Conclusion Bortezomib can inhibit primary leukemia cells from elderly patients proliferation and induce apoptosis.The mechanism may be associated with the changes in bcl-2 family protein expression.

KLD-12 peptide with a sequence of AcN-KLDLKLDLKLDL-CNH(2) was synthesized and its biocompatibility was assessed in animals. Rabbit MSCs were cultured in the hydrogel for 2 weeks. Live cells were counted by using Calcein-AM/PI fluorescence staining. MTT was employed to assess the viability of MSCs cultured in KLD-12 peptide solution of 0.01%, 0.03%, and 0.05%. Hemolysis test, skin irritation test and implantation test were conducted to evaluate its biocompatibility with host tissues. Our results demonstrated that the MSCs in hydrogel grew well and maintained round shape. Cell survival rate was 92.15% (mean: 92.15%+/-1.17%) at the 7th day and there was no difference in survival rate between day 7 and day 14. Cell proliferation test showed that the A value of the KLD-12 solutions was not significantly different from that of control groups (complete culture media) (P>0.05) at the 24th and 48th h. The hemolysis rate of KLD-12 solution was 0.112%. Skin irritation test showed that the skin injected with KLD-12 solution remained normal and the score of skin irritation was 0. The histological examination with HE staining exhibited that the skin layers were clear and there was no infiltration with neutrophilic granulocytes and lymphocytes. It is concluded that KLD-12 peptide hydrogel had a good biocompatibility with host rabbit and MSCs, and KLD-12 peptide hydrogel can provide an appropriate microenvironment for MSCs.

o-fiber hydrogel in vitro. MSCs in KLD-12 peptide hydrogel grew well and proliferated with the culture time. KLD-12 peptide hydrogel can serve as an excellent injectable material of biological scaffolds in tissue engineering of IVD.

OBJECTIVETo investigate the variations of HCV core and E2 region epitopes during transfusion of activated immune cells.

METHODSFour patients receiving transfusion of activated immune cells were under continuously observation. HCV titers were measured by quantitive PCR. HCV core and E2 regions were cloned and sequences were analyzed by computer software.

RESULTSDuring the follow-up the serum ALT levels and the HCV virus titers fluctuated greatly in each of these persons. After transfusion, no significant variations were observed in HCV core and E2 coding regions.

CONCLUSIONSThe alteration of host immune attacks could induce the fluctuations of HCV load without any mutations in the currently observed coding genes of the epitopes.

Adult ; Aged ; Alanine Transaminase ; blood ; Epitopes ; genetics ; Female ; Genetic Variation ; Hepacivirus ; genetics ; Hepatitis C, Chronic ; blood ; therapy ; virology ; Humans ; Immunotherapy ; Male ; Middle Aged ; Viral Core Proteins ; genetics ; Viral Envelope Proteins ; genetics ; Viral Load

KLD-12 peptide with a sequence of AcN-KLDLKLDLKLDL-CNH2 was synthesized and its biocompatibility was assessed in animals.Rabbit MSCs were cultured in the hydrogel for 2 weeks.Live cells were counted by using Caicein-AM/PI fluorescence staining.MTT was employed to assess the viability of MSCs cultured in KLD-12 peptide solution of 0.01%,0.03%,and 0.05%.Hemolysis test,skin irritation test and implantation test were conducted to evaluate its biocompatibility with host tissues.Our results demonstrated that the MSCs in hydrogel grew well and maintained round shape.Cell survival rate was 92.15%(mean: 92.15%±1.17%)at the 7th day and there was no difference in survival rate between day 7 and day 14.Cell proliferation test showed that the A value of the KLD-12solutions was not significantly different from that of control groups(complete culture media)(P>0.05)at the 24th and 48th h.The hemolysis rate of KLD-12 solution was 0.112%.Skin irritation test showed that the skin injected with KLD-12 solution remained normal and the score of skin irritation was 0.The histological examination with HE staining exhibited that the skin layers were clear and there was no infiltration with neutrophilic granulocytes and lymphocytes.It is concluded that KLD-12 peptide hydrogel had a good biocompatibility with host rabbit and MSCs,and KLD-12 peptide hydrogel can provide an appropriate microenvironment for MSCs.