ABSTRACT
Cardiac stem cells (CSCs) transplantation has been recognized to be effective on the treatment of myocardial infarction (MI), but some techniques still need to be developed in the isolation and culture of CSCs, which is the key problem restricting the clinical application of CSCs. This study was focused on the isolation of Lin(lineage-negative) Sca-1(stem cell antigen-1-positive) CSCs from newborn C57BL/6J mice (0-3 d) by mixed enzymatic-explant isolation in combination with immunomagnetic separation. The digesting time, digesting frequency, incubation temperature, stirring speed, centrifugation time and rotational speed were strictly controlled in the experiment. In order to increase the survival rate of CSCs, the medium changing time and manner were optimized in primary CSCs culture. The percentages of Sca-1cells in primary and passage cells were detected by flow cytometry and immunofluorescence staining. The results showed that: (1) the proportion of LinSca-1cells within the collected cells could be as high as (85.03 ± 5.60)% after isolation and purification; (2) In vitro culture of LinSca-1CSCs grew into spheres on the 5day, and over the whole bottom of the dish on the 7day. The growth curve showed that the cells were in logarithmic growth phase on the 3day; (3) Immunofluorescence staining data showed that the expression of Sca-1, the CSCs membrane-specific marker, was decreased after subculture, and flow cytometry data showed that the percentages of Sca-1cells were (71.82 ± 2.63)%, (58.38 ± 3.70)% and (46.19 ± 4.72)% in passage 1 (P1), P3, and P5 CSCs, respectively. The above results suggest that high purity of LinSca-1CSCs can be obtained by enzymolysis combined with immunomagnetic separation method. Moreover, the CSCs culture system is stable. In our experiment, the Sca-1CSCs isolation and culture method has been successfully established, and it is simple, stable, effective and reliable. The method can provide a stable methodological basis for the treatment of MI by LinSca-1CSCs transplantation.
ABSTRACT
Objective To prospectively assess the predictive power of centralization phenomenon in the curative effect of automated PLD.Methods The survey population was consisted of 109 patients with inclusion heraiation demonstrated by CT/MRI,74 men and 35 women with average age of 43.1 years(17～75 years). All were complained of low back pain,with varying degrees of lower extremity pain and altered sensation, lasting for more than 2 months;including one symptomatic disc in 99 patients and two symptomatic discs in 10 patients.Patients were undergone dynamic mechanical spinal test and reported whether the test would aggravate their pain.The assessment included forward flexion,extension,rotation of the trunk to the right and left, rotation to the left with fight extension,rotation to the fight with left extension,and whether straight leg raising in the supine position would aggravate back pain or leg pain.Symptom resposes were categorized into three groups:centralization group(CG),partial-centralization group(PCG)and noncentralization group(NCG). Centralization of pain is the progressive retreat of the most distal extent of the referred or radicular pain toward or to the lumbar midline.Noncentralization of pain is the peripheralization of pain in one or more directions, and no change in the distal-most pain location or intensity.All patients received a single therapy with PLD. Results A follow-up of 109 patients for 3 to 6 months,including 46 cases with 24 as exellent and 22 as good reaching 100% of excellent good rate in CG by MacNab standards;43 cases with 5 as exellent,29 as good,9 as fair and poor,with total effective rate of 79.1% in PCG.Twenty cases of NCG symptoms showed no improvement and therefore surgery was considered.Conclusions Centralization phenomenon occurrence during initial mechanical evaluation is a very accurate predictor for successful PLD outcome.Nonoccurrence of centralization would accurately predict poor PLD outcome and thus helpful as early predictor of the need for surgical treatment.