[Targeted delivery of bone marrow mesenchymal stem cells by ultrasound-mediated microbubble destruction].

OBJECTIVE: To investigate the feasibility of bone marrow mesenchymal stem cell (MSC) transplantation with ultrasound-targeted microbubble destruction. METHODS: Twenty-one Wistar rats were divided into MSCs-iv group (MSCs-iv), ultrasound+MSCs-iv group (US+MSCs-iv), ultrasound+microbubble+MSCs-iv group (US+MB+MSCs-iv) with intravenous MSC transfer, ultrasound and microbubble treatment as indicated. The skeletal muscles were obtained from the rats for microscopic examination with HE staining. The hindlimb gracilis and semimembranosus muscles were sampled 7 days after MSC transplantation, and the transplanted MSCs were detected by immunohistochemistry. The vital organs were collected from rats in US+MB+MSCs-iv group for immunohistochemistry. RESULTS: In US+MB+MSCs-iv group, HE staining demonstrated the presence of red blood cell leakage into the tissue space in the gracilis and semimembranosus muscles, and immunohistochemistry identified large numbers of transplanted MSCs in the the gracilis and semimembranosus muscles and the spleen, whereas no labeled cells were detected in the skeletal muscles in other groups. CONCLUSION: Ultrasound-targeted microbubble destruction provides a useful means for enhancing the efficiency of stem cell transplantation.

[Assessment of direct effects of dobutamine on coronary microcirculation with myocardial contrast echocardiography: comparison with adenosine].

OBJECTIVE: To evaluate the direct effects of dobutamine as compared to adenosine on the coronary microcirculation in both normal and stenotic segments using myocardial contrast echocardiography (MCE). METHODS: Left anterior descending (LAD) coronary artery stenosis, which was not flow limiting at rest, was established in 9 dogs. At the baseline and during intracoronary infusion of dobutamine (2 mg.kg(-1).min(-1)) and adenosine (5 mg.kg(-1).min(-1)), the radiolabeled microsphere (RM)-derived myocardial blood flow (MBF) were determined, and the double product (DP) and myocardial vascular resistance (MVR) were calculated. MCE was performed to determine the myocardial blood volume (MBV, represented by A) and microbubble velocity (beta). RESULTS: As compared to the baseline level, the MBF increased and MVR decreased significantly in both the normal and abnormal beds during infusion of both drugs (P<0.05). In the normal bed, adenosine had no effect on MBV, the decrease in MVR was the result of decreased arteriolar (plus venular) resistance, and the increase in MBF was predominately due to the increase in b (deltabeta/ deltaA=13.6). Dobutamine caused a 28% increase in MBV, responsible for 32% of the decrease in the total MVR, but the increase in MBF arose mainly from the increase in b (deltabeta/deltaA=5.9). In the abnormal bed, both the drugs caused a similar increase in MBF entirely by increasing b, and 14% and 15% of the increases in capillary resistance were associated with the capillary derecruitment during administration of dobutamine and adenosine, respectively. CONCLUSION: The direct effects of intracoronary dobutamine infusion on the coronary microcirculation are similar to that of adenosine, and the increase in MBF occurs mostly as the result of increased myocardial blood velocity.

[Cloning of expression vector for VEGF121 and VEGF165 genes encoding human vascular endothelial growth factor].

OBJECTIVE: To clone and construct the expression vector for human vascular endothelial growth factor (VEGF) genes. METHODS: Total RNAs were extracted from human lung tissue of a 4-month-old fetus and subjected to reverse transcriptase-polymerase chain reaction (RT-PCR) with the amplified products cloned into pMD18-T vector. Sequence analysis was performed before the amplified products were cloned into the expression plasmid pcDNA3.1-, the recombinant of which was verified by endonuclease digestion. RESULTS: After RT-PCR using a pair of primers (sense -21/7 bp and antisense 554/576 bp), two bands were identified. The band (487 bp) shorter in length was confirmed as VEGF121 (with the full length of VEGF121 being 444 bp) while the longer band (619 bp) was normal VEGF165 (with the full length of VEGF165 being 576 bp). Interestingly, another slightly longer VEGF165 nucleotide sequence was identified by sequencing analysis, which featured an unique 20 bp insertion precisely between exon 3 and exon 4 from the first ATG of human VEGF165 cDNA. The 20 bp insert was identified as the retaining intron 3 terminal nucleotides containing the splicing signal, which caused frame shift mutation in the reading frame and could probably give rise to a short polypeptides consisting of only 97 amino acid residuals due to the early appearance of stop code UAG in the middle of exon 4. CONCLUSION: We have successfully constructed the expression vector for VEGF121 and VEGF165 genes, and a new possible alternative splicing isoform of VEGF is identified in normal fetus whose molecular mechanism and physiological function needs further investigation.