Therapeutic effects of CXCR4+ subpopulation of transgene-free induced cardiosphere-derived cells on experimental myocardial infarction.

OBJECTIVES: Myocardial infarction (MI) is the most predominant type of cardiovascular diseases with high mortality and morbidity. Stem cell therapy, especially cardiac progenitor cell therapy, has been proposed as a promising approach for cardiac regeneration and MI treatment. Previously, we have successfully generated cardiac progenitor-like cells, induced cardiosphere (iCS), via somatic reprogramming. However, the genome integration characteristic of virus-based reprogramming approach hampered their therapeutic applications due to the risk of tumour formation. In the current study, we aim to establish a safer iCS generation strategy with transgene-free approaches. MATERIALS AND METHODS: Four transgene-free approaches for somatic reprogramming, including episome, minicircle, self-replicative RNA, and sendai virus, were compared, from the perspective of cardiac progenitor marker expression, iCS formation, and cardiac differentiation. The therapeutic effects were assessed in the mouse model of MI, from the perspective of survival rate, cardiac function, and structural alterations. RESULTS: The self-replicative RNA approach produced more iCS, which had cardiomyocyte differentiation ability and therapeutic effects on the mouse model of MI with comparable levels with endogenous cardiospheres and iCS generated with retrovirus. In addition, the CXCR4 (C-X-C chemokine receptor 4) positive subpopulation of iCS derived cells (iCSDC) delivered by intravenous injection was found to have similar therapeutic effects with intramyocardial injection on the mouse model of MI, representing a safer delivery approach. CONCLUSION: Thus, the optimized strategy for iCS generation is safer and has more therapeutic potentials.

A disposable fiber optic SPR probe for immunoassay.

Immunoassay can be divided into two aspects, one is immobilization of antibodies for the efficient detection of corresponding antigens, and the other is immobilization of antigens to search for antibodies which work against them. In this paper, we demonstrated these two aspects of immunoassay by using the disposable fiber optic biosensors based on surface plasmon resonance (SPR) through surface decoration with half-antibody fragments, which has been scarcely ever reported to the best of our knowledge. We first fabricated the fiber optic SPR biosensor which consists of one gold film coated single-mode fiber sandwiched by two multimode fibers. Then, we decorated the fiber optic SPR biosensors with antibody fragments and antigen fragments, respectively, and compared the specific detection performances of these two kinds of sensors. After surface decoration with half-antibody fragments, the antigen-decorated fiber probe has a demonstrated sensitivity and limit of detection of 0.9771nm/(µg/mL) and 0.1 µg/mL, respectively, which improves by 10 times compared with the performance of the antibody-decorated fiber probe. Additionally, the selective detection results indicate that our proposed biosensor can be employed as a reliable antigen detector or an effective antibody filter. Our proposed sensor has the advantages of miniaturization, low cost, simple usage, label-free detection, high efficiency and sensitivity, and can effectively avoid cross-contamination caused by reuse. Given the reliable and clean detection method for immunoassay, our work should open a new window for the utilization of miniaturized fiber optic sensors in biochemical sensing.

A label-free fiber optic SPR biosensor for specific detection of C-reactive protein.

A highly sensitive and label-free fiber optic surface plasmon resonance (SPR) biosensor for specific detection of C-reactive protein (CRP) is proposed and demonstrated. We take dopamine as a cross-linking agent to immobilize the anti-CRP monoclonal antibody, which is an efficient and simple method for specific modification of the fiber optic SPR sensor. The modified sensor can successfully detect CRP specifically. We realize the fabrication of a disposable fiber optic SPR sensor for the CRP specific detection. Through optimizing the immobilization time of anti-CRP monoclonal antibody and the reaction time of antigen and antibody experimentally, the sensor shows a satisfactory linear response (R2 = 0.97) to CRP concentration within the range from 0.01 to 20 µg/ml. Moreover, the highest CRP sensitivity is obtained at 1.17 nm per lg (µg/ml). With the advantages of simple structure and easy fabrication, our sensor is convenient to be batch produced and controlled with good consistency, which is especially suitable for the fabrication of disposable biosensor. It makes sense that our detection can effectively avoid the cross pollution caused by repeated use of the sensor.

[Establishment of the human papillomavirus type 31 positive cervical cancer cell line].

The establishment of in vitro model will provide optimal conditions for the study of human papillomavirus (HPV)-associated cervical cancer. In this study, E6 and E7 gens of HPV31 were cloned and expressed in E. coli. The recombinant proteins were purified and used as antigens to immunize mice for the production of polyclonal antibody. Mammalian expression plasmid pBudCE4. 1-HPV31-E6/E7 was also constructed and transfected into C33A cells. The transfected cells were then selected by Zeocin. The expressions of the E6 and E7 mRNAs and proteins were detected by RT-PCR and Western blot respectively. A stable cervical cancer cell line was established as an in vitro model for the study of human papillomavirus type 31(HPV31) associated cervical cancer.

[Expression, purification and antigenicity of P35 gene of Toxoplasma gondii].

OBJECTIVE: P35 is an important surface protein for Toxoplasma gondii. To obtain the highly pure and specific antigenicity protein, the gene P35 was cloned and its product was expressed in E. coli Rosetta. The expressed protein was purified and its immunogenecity was studied. METHODS: A pair of primers was designed according to cDNA sequence of P35, and then the P35 gene amplified by PCR was cloned into the vector pGEM-T and proved by DNA sequencing. The P35 gene was subcloned into prokaryotic expression vector pET-KDO, its expression was induced by IPTG, and the target protein was obtained by affinity chromatography. RESULTS: The P35 gene was successfully amplified from genome DNA of Toxoplasma gondii RH strain and a fusion protein was expressed in E. coli. The molecular weight of the expressed protein was about Mr 42 000. Western blotting indicated that the antigenicity of the protein was specific. CONCLUSIONS: The plasmid pET-KDO-p35 is constructed and the high efficient expression of P35 fusion protein has been achieved in E. coli. The fusion protein shows a specific antigenicity, the P35 fusion protein has a potential value in the diagnosis of toxoplasmosis.