Isolation And Study On The Pulmonary Surfactant From Hominine Lung

Pulmonary surfactant (PS) is a lipoprotein complex mainly consisting of phospholipids and specific proteins. Down-regulation of PS plays a important role in the development of respiratory distress syndrome (RDS), resulting in a high morbidity and mortality, particularly in preterm infants. Administration of exogenous PS is a effective method for treating RDS and also used in other pulmonary diseases.At present, there is only Two kinds of PS preparation. The first one is Curosurf, manufactured by Kaisi Pharmacy Company in Italy, was approved to treat neonatal respiratory distress syndrome (NRDS) in our country. The price of it is very high, 4500 RMB per ampoule. The other PS reagents that developed by other domestic institutes were just got the certificate from SFDA in Nov, 2005. The price of it is 3750 RMB per ampoule. In Coal-workers sanitarium Chen Zhiyuan has cured over 2000 silicosis patients with Whole-Lung Lavage(WLL).These experiments extracted Pulmonary Surfactant from silicosis patients lungs. And it is believed that Pulmonary Surfactant from hominine lungs would be better ,cheaper than Curosurf and other PS reagents from animals'lungs,and more conformable used in babies. Objective To explore the contents of Surfactant Protein A and Phospholipid in Whole-Lung Lavage (WLL) liquid of Silicosis Patients. Methods The 6 term-Ⅰand 6 term-ⅡSilicosis patients were researched according to x-ray diagnosis. The Surfactant Protein A (SP-A) and Phospholipid (PL) in the WLL liquid were extracted and measured with ELISA and the photometer. Results The contents of SP-A in WLL liquid in term-Ⅰand term-ⅡSilicosis group were 87.21±17.23mg and 56.97±13.44mg respectively. The contents of PL were 222.92±26.04mg, 121.31±29.62mg respectively. The contents of SP-A, PL in term- I group were higher than those in term-Ⅱgroup, p＜0.01. Conclusions The increase of SP-A/PL in WLL liquid may be effective biomarkers for early Silicosis. And it was believed that SP-A/PL could be mass-extracted in the patients with early Silicosis.Objective The pulmonary surfactant (PS) is used to cure neonatal respiratory distress syndrome (NRDS) , acute respiratory distress syndrome (ARDS), and acute lung injury (ALI) so on. By far there has not been a convenient technique to mass-produce PS in the huge volume of derelict Whole-Lung Lavage (WLL) liquid of Silicosis Patients. Thus it is urgent to find a applied technique. Methods 9 experiments were designed with the Orthogonal Oxperiment Design and finished in 3 levels and 4 factors, which were respectively the first centrifugal rotate speeds in 1500, 2000, and 3000 g, the dialytic molecular apertures in 3000, 10000, and 14000 dalton, the second centrifugal rotate speeds in 5000, 10000, and 15000 g, and the extractive distillation with chloroform-carbinol, aether or n-butanol. At last the best technique was choosed according to output of PS. Results The best technique is that, the first centrifugal rotate speeds is 3000g, the dialytic molecular apertures is 3000 dalton, the second centrifugal rotate speeds is 10000 g, and the extractive distillation is with n-butanol. With the way the raw product from 6 men had gotten to 7968.6222mg o Conclusions It was believed that the best technique could competently be used in mass-production on PS in the huge volume of derelict Whole-Lung Lavage (WLL) liquid of Silicosis Patients.Objective To get the purifying Pulmonary Surfactant from the primary production. Method Second-purified Pulmonary Surfactant with the Sephrose Chromatogram. Result HPS was got to 12646.76 mg from 8 men.The percentage of Pulmonary Surfactant in terminal production is got to 98%. Conclusion It is feasible to purify Pulmonary Surfactant with the Sephrose Chromatogram. Objective To study second-purified Pulmonary Surfactant and compare with other reagents. Methods Analytical chemistry on Pulmonary Surfactant detect the content of lecithoid, protein, cholesterin and triglyceride. The component of Pulmonary Surfactant is detected with High-powered liquid Chromatogram. Results There is 1580.85mg in one patient's WLL liquid. In it the lecithoid is 95.74%, protein 2.15%, cholesterin 1.02%, triglyceride 1.09%. And in the lecithoid the phosphatidyl choline is 80.43% , phosphatidylglycerol 6.67% , phosphatidylethanolamine 6.78%, phosphatidylinositol 3.77%, sphingomyelin 2.35%. Conclusion The second-purified Pulmonary Surfactant is suitable to use as a reagent.Objective To extract Pulmonary Surfactant A from derelict Whole-Lung Lavage (WLL) liquid of Silicosis Patients. Methods Purify Pulmonary Surfactant A from the primary production with the gelose-gel Chromatogram, and identify it with ELISA. Results The content of SP-A is 1.3692g, which is 92.20% in extracted production. Conclusion It is feasible to extract Pulmonary Surfactant A with Extraction, Dialysis and Chromatogram in the derelict Whole-Lung Lavage (WLL) liquid of Silicosis Patients. Objective To determine Effectiveness of Pulmonary Surfactant in Vivo. Methods PS and SP-A were put on distilled water and determine Effectiveness of them in Vivo with JM04 dynamic tension recorder. Results The surface tension of distilled water is 14.16±2.07 mN/m in 20% of quondam landmeasure to 1.13±0.40mN/m in 100% of quondam landmeasure. The surface tension of 50 ul PS in distilled water is 21.38±2.20 mN/m to 0.74±0.08 mN/m. And the surface tension of 50 ul PS+SP-A in distilled water is 25.67±2.91mN/m to 0.51±0.17mN/m.Statistical test showes P＜0.001 among the 3 groups. Conclusions It is believed that Pulmonary Surfactant in Vivo could stabilify the surface tension of distilled water. And Pulmonary Surfactant A is helpful to make PS extend on the surface of distilled water.Objective To illuminate contents of PS in Patients with asynchronous Silicosis. Methods PS and SP-A were extracted with forenamed technology. Then contrasted their contents. Results Content of PS in the patient with Pulmonary Alveolar Proteinosis is 287.4mg/1, SP-A is 2.89g.The content of PS in Patients with I term Silicosis is 137.13±17.40mg/1, SP-A 0.95±0.20g.PS inⅡterm Silicosis is 32.6±3.89mg/l, SP-A 0.61±0.16g, PS inⅢterm 29.9±6.43mg/1, SP-A 0.52±0.23g. Statistical test showes P＜0.001 among the 3 groups.Conclusions It is believed that content of PS and SP-A in the patient with Pulmonary Alveolar Proteinosis is more than all of the patients with Silicosis. In them, PS inⅠterm patients is more thanⅡ,Ⅲterm, and SP-A in the same way.Objective To research compensative capacity of PS Level after Whole-Lung Lavage (WLL) on patients with Silicosis. Methods WLL was used to cure Silicosis patients. After that their lungs were lavaged at 0, 10th, 30th minute again. PS were extracted in succedent liquid with forenamed technology. Then contrasted their levels. Results After WLL the levels of PS are 263.4±20.37mg/l, 183.8±19.34mg/l, 77.7±1.94mg/l inⅠ,Ⅱ,Ⅲterm Silicosis patients respectively.10 minutes later the levels are 460.4±22.03mg/l, 191.2±18.9mg/l, 77.9±01.82mg/l respectively. 30 minutes later the levels of PS are 842.8±34.17mg/l,425.8±20.04mg/l, 81.8±3.40mg/l. Statistical test showes P＜0.001 among the 3 groups.Conclusions It is believed thatⅠ,Ⅱterm patients lungs own great compensative capacity. PS inⅠterm patients lungs could come back in 10minutes.PS inⅡterm patients" lungs could come back in 30minutes. PS inⅢterm patients" lungs could not come back in 30minutes in nature.Summary1 The increase of SP-A/PL in WLL liquid may be effective biomarkers for early silicosis. And it was believed that SP-A/PL could be mass-extracted in the patients with early silicosis.The contents of SP-A in WLL liquid in term-Ⅰand term-ⅡSilicosis group were 87.21±17.23mg and 56.97±13.44mg respectively. The contents of PL were 222.92±26.04mg, 121.31±29.62mg respectively. The contents of SP-A/PL in term-Ⅰgroup were higher than those in term-Ⅱgroup.2 The best technique is that, the first centrifugal rotate speeds is 3000g, the dialytic molecular apertures is 3000 dalton, the second centrifugal rotate speeds is 10000g, and the extractive distillation is with n-butanol. With the way the raw product from 6 men had gotten to 7968.6222mg。3 It is feasible to purify Pulmonary Surfactant with the Sephrose Chromatogram. HPS was got to 12646.76 mg from 8 men.The percentage of Pulmonary Surfactant in terminal production is got to 98%.4 The second-purified Pulmonary Surfactant is suitable to use as a reagent. There is 1580.85mg in one patient's WLL liquid. In it the lecithoid is 95.74%, protein 2.15%, cholesterin 1.02%, triglyceride 1.09%. And in the lecithoid the phosphatidyl choline is 80.43%, phosphatidylglycero16.67%, phosphatidylethanolamine6.78%, phosphatidylinositol 3.77%, sphingomyelin 2.35%.5 The content of SP-A is 1.3692g, which is 92.20% in extracted production. It is feasible to extract Pulmonary Surfactant A with Extraction, Dialysis and Chromatogram in the derelict Whole-Lung Lavage (WLL) liquid of Silicosis Patients.6 The surface tension of distilled water is 14.16±2.07mN/m in 20% of quondam landmeasure to 1.13±0.40mN/m in 100% of quondam landmeasure. The surface tension of 50 ul PS in distilled water is 21.38±2.20mN/m to 0.74±0.08mN/m. And the surface tension of 50 ul PS+SP-A in distilled water is 25.67±2.91mN/m to 0.51±0.17mN/m. It is believed that Pulmonary Surfactant in Vivo could stabilify the surface tension of distilled water. And Pulmonary Surfactant A is helpful to make PS extend on the surface of distilled water.7 It is believed that content of HPS in t patient with Pulmonary Alveolar Proteinosis is more than all of the patients with Silicosis. SP-A is also. In them, HPS inⅠ,Ⅲterm patients is more thanⅡterm, and SP-A inⅠ,Ⅱterm patients is more thanⅢterm.Content of HPS in the patient with Pulmonary Alveolar Proteinosis is 287.4mg/L, SP-A is 2.89g.The content of HPS in Patients with I term Silicosis is 137.13±17.40mg/L, SP-A 0.95+0.20g.PS inⅡterm Silicosis is 32.6±3.89mg/L, SP-A 0.61±0.16g, PS inⅢ29.9±6.43mg/L, SP-A 0.52±0.23g.8 It is believed thatⅠ,Ⅱterm patients" lungs own great compensative capacity. PS inⅠterm patients lungs could come back in 10minutes.PS inⅡterm patients lungs could come back in 30minutes. PS inⅢterm patients lungs could not come back in 30minutes in nature.